In recent years, buspirone has been co-administered with sertraline to resolve sexual disorders caused by sertraline. Therefore, the present study was conducted to investigate the interaction effect of two antidepressants and anxiolytic drugs, sertraline and buspirone, on human serum albumin (HSA) using spectroscopic and molecular docking techniques. Fluorescence emission spectroscopy and molecular docking were used to calculate the binding affinity and determine the best binding sites for these two drugs. Additionally, UV-visible and circular dichroism spectroscopy were performed to investigate the effect of these drugs on the conformational changes of HSA. The results showed that both drugs have a strong ability to quench the fluorescence of HSA through a static mechanism, and cause structural changes in HSA. It was also found that binding of sertraline and buspirone to HSA is spontaneous and hydrophobic interactions, van der Waals forces and hydrogen bonds play a significant role in these interactions in the ternary system. In addition, molecular docking data showed that both drugs bind with high affinity to the Trp residue in subdomain IIA. This study demonstrates that the presence of the second drug (buspirone/sertraline) affects the interaction and binding affinity of the first drug (sertraline/buspirone) to human serum albumin.