BACKGROUND: Active surveillance testing (AST) is one element of a comprehensive Carbapenem-resistant Enterobacteriaceae (CRE) prevention strategy. However, the utility of AST may be impacted by compliance with sample collection and the quality of specimens. Here, we describe strategies used to optimize a CRE AST program at a large academic medical center. METHODS: Tests ordered, collected, rejected, and processed were tracked weekly for each participating unit. Sample collection compliance and acceptance rates were calculated and tracked weekly. Strategies were implemented to improve collection compliance and sample acceptance rates, including computerized provider order entry, printed educational materials, and audit and feedback. Weekly dedicated Infection Preventionist (IP) time was estimated. RESULTS: Over 35 months, mean collection compliance increased from 82.7% to 91.2%, and then decreased to 86.2%. Over 27 months, sample acceptance rate increased from 57.7% to 83.6%, and then remained stable at 83.4%. Over 39 months, dedicated weekly IP time decreased 92%. DISCUSSION: Use of evidence-based quality improvement strategies optimized our CRE AST program. Optimizing the AST process aids in early CRE detection, leading to timely isolation and preventing the spread of CRE to other patients. Other hospitals may benefit from these lessons and enhance local AST programs.