Background: The modern practice of clinical chemistry relies ever more heavily on automation. Their utilization in clinical laboratories of developing countries is greatly affected by many factors. Thus, identifying the different challenges relating to clinical chemistry automation utilization faced by laboratories is important to work on and resolve. Method: Cross-sectional study was conducted in 15 public hospitals found in Southwest Shoa, Jimma, Ilubabor and Buno-Bedele zones of Oromia region, Ethiopia from January 28 to March 15, 2019. Sixty eight key informants and ninety three laboratorians who were working in the clinical chemistry section were included in the study. Data were collected by self-administered questionnaires, indepth interviews and observation. The quantitative data were analyzed by simple descriptive statistics using SPSS 25.0 whereas qualitative data were analyzed manually. Results: There were 14 different models of clinical chemistry analyzers. More than two-thirds of analyzers were out-of-service. In another way, only 14 (15.1%) of the laboratorians had received user training of clinical chemistry analyzers. Majority of the laboratories were suffered from clinical chemistry reagents shortage. There were also inappropriate procurement processes of the clinical chemistry analyzers, misuse and underuse of clinical chemistry tests. Conclusion: The survey and interviews specified six major challenges that seriously obstruct utilization of clinical chemistry analyzers. Keywords: Challenges; analyzers utilization; clinical chemistry analyzers; automation.