Background: The incidence rates of endoscopy use associated healthcare-associated infection were reported to be 1 in 1.8 million procedures, but contaminated endoscopes might cause outbreaks more often than other medical devices. Aim: The aim of this study was to describe the endoscope reprocessing procedure validated with microbiological cultures and ATP bioluminescence tool kits. Material and methods: We examined the endoscope reprocessing procedures at the Ministry of Health Bakirkoy Sadi Konuk Research Hospital using microbiological cultures and ATP bioluminescence tool kit during 2014. Results: We examined 42 flexible endoscopes and 4 (9%) endoscopes were found to be contaminated. Stenotrophomonas maltophilia (>100,000 cfu/mL) was isolated from irrigation bottles and extended-spectrum beta-lactamases and plasmid-mediated carbapenamase producing K. pneumonia and AmpC beta-lactamase producing P. aeruginosa (>100,000 cfu/mL) were isolated from elevator behind, respectively. Each step of the reprocessing procedure has been examined and revised with microbiological cultures and ATP bioluminescence tool kit. All endoscopes were found to be clean after revising the procedure. Conclusion: ATP-bioluminescence method provides shorter time to examine the endoscopes as a cost-effective method in the endoscope reprocessing. Microbiological monitoring and cleaning procedures of endoscopes should be defined in the healthcare settings with the frequencies. In case of any outbreak related to endoscopes, each step of procedure should be examined and corrected taking into guidelines, reported outbreaks, as well as instructions of endoscope manufacturers.