While various methods exist for the detection of Mycoplasma contamination [13, 14], probably the most frequently used ones are the biochemical detection of Mycoplasma metabolism and PCR-based detection of Mycoplasma DNA. Though the biochemical detection of mycoplasmal ATP generation (Mycoalert (Lonza, Basel, Switzerland)) is a quick protocol, it has certain disadvantages that should be mentioned, including the requirement of the reagents to be reconstituted and be brought to 22oC before each measurement, and the availability of a luminometer for ATP detection. Aspecificity due to ATP generated by other cells may lead to a high background and eventually false negative measurements. The Ureaplasma species which are also a common contaminant in a cell culture  cannot be detected by Mycoalert as their own ATP production relies on the hydrolysis of urea . Lastly, the sensitivity of the biochemical detection was shown to be lower than that for PCR or qPCR methods [17, 18]. There is a variety of kits on offer based on regular PCR, followed by gel electrophoresis. The major advantage of these kits is the wide availability of regular PCR and electrophoresis equipments. However, the decreased specificity compared to a probe-based qPCR, the additional electrophoresis step, and the lack of quantitative monitoring of the decrease in the Mycoplasma genome concentration during treatment are clear drawbacks. The intercalation-based (e.g. SYBR Green) qPCR kits such as MycoSEQ Mycoplasma Detection Assay (Thermo Fisher, Waltham, MA, USA) eliminate the electrophoresis step and provide quantitative information about the Mycoplasma genome concentration. The disadvantages of intercalation-based qPCR kits compared with the probe-based kits are the lower specificity, the lack of internal control and the potential effect of the cell culture composition, ionic composition and ionic strength on the melting temperature of the qPCR product [19–21]. Since the melting temperature is the basis for evaluating specificity in the intercalation based qPCRs, shifting the melting temperature in a direct qPCR can be a problem. The probe-based qPCRs such as PhoenixDx (Procomcure Biotech, Thalgau, Austria), Microsart RESEARCH Mycoplasma (Sartorius, Goettingen, Germany) and the qPCR Detection Kit (XpressBio, Frederick, MD, USA) avoid these problems and due the additional requirement of the binding of the probe sequence, these kits provide a higher specificity than the regular PCRs and intercalation-based qPCRs. Noting the above-mentioned advantages of probe-based qPCRs, we optimized the Procomcure PhoenixDx kit to perform a direct qPCR with a Mycoplasma infected U937 cell culture. Our results indicates that the optimal temperature was the same as that in the original protocol, so the primer+probe binding was not affected by the presence of the direct template. The fact that the optimal template volume was 6 l (30% of the total qPCR volume) meant that the direct sample did not have a significant inhibitory effect on the qPCR. A major optimization step that we performed decreased the annealing/extension time from 60 sec to 20 sec, thus saving 40 seconds in each cycles. Interestingly, this decrease led to only a minor decrease in the sensitivity (~0.6 Ct level increase). In addition to decreasing the cycle number from 50 to 40, the total qPCR time required was reduced to 65 minutes. When we used the optimized qPCR protocol with direct and purified cell culture templates, we found that the Ct levels of a 6 l direct template was almost identical with that of the purified DNA from a 60 l cell culture. The reason for this is mainly due to a dilution of the original DNA content during the elution step at the end of the DNA purification. Overall in our case the direct qPCR sensitivity was higher than the qPCR with a purified template, with a saving in the cost/time of DNA purification. Then we followed the elimination of Mycoplasma contamination from the U937 cell culture using the optimized direct qPCR protocol. One of the concerns using pathogen DNA detection is that the non-viable pathogen’s DNA can also be detected and lead to a false positive signal. In our case, however, the Mycoplasma DNA content dropped to ~20% of the original concentration after 1 day of treatment, and though days 1 and 2 contained a similar level of DNA, this decrease continued on day 3. In summary, with direct qPCR we were able to monitor the elimination of Mycoplasma over the treatment period. In conclusion, we optimized a probe-based qPCR to detect Mycoplasma contamination in a user-friendly manner. This direct qPCR method does not require a purification step, it maintains sensitivity and it offers a shorter 65-minutes protocol. Limitations While we did not observe a major qPCR inhibitory effect of U937 cell culture, it cannot be ruled out that components of other cell cultures may have an inhibitory effect. Most probe based qPCR kits, including the kit used here, contains an internal control (e.g. HEX-labeled probe), therefore the detection of qPCR inhibition (no FAM, no HEX signals) is straightforward. In the case of qPCR inhibition, dilution of the direct sample may be a solution for decreasing/eliminating qPCR inhibition.