In this prospective study all Enterobacteriaceae isolates (n=2129) recovered in the clinical microbiology laboratory during October 2009 to April 2010 were analyzed for AmpC production. CLSI cefoxitin and cefotetan susceptibility breakpoints and CLSI critical ESBL diameters were used to screen for potential AmpC producers. In total, 305 isolates (211 potential AmpC producers, 94 AmpC screen negative isolates as control group) were further analyzed by multiplex PCR for the detection of plasmid-encoded ampC beta-lactamase genes and by ampC promoter sequence analysis (considered as gold standard). Cefoxitin and cefotetan were assessed as primary screening markers. Sensitivities of cefoxitin and cefotetan for detection of AmpC production were 97.4% and 52.6%, specificities were 78.7% and 99.3%, respectively. As a phenotypic confirmation test, the Etest AmpC and the cefoxitin/cloxacillin double disk synergy method (CC-DDS) were compared. Sensitivities for the Etest AmpC and the CC-DDS method were 77.4% and 97.2, respectively; specificity was 100% for both methods. The results of the Etest AmpC were inconclusive for 10 isolates. With the CC-DDS method 2 inconclusive results were observed. Based on this study we propose a comprehensive diagnostic flow-chart for detection of AmpC production consisting of a simple phenotypic screening and a single phenotypic confirmation test with inconclusive results being resolved by molecular analysis. For the proposed flow-chart using i) cefoxitin as screening marker for AmpC production ii) the CC-DDS method as phenotypic confirmation and iii) molecular methods in case of inconclusive results, the sensitivity and specificity for AmpC detection would have been 97.4 and 100%, respectively, with respect to the studied isolates. The phenotypic methods used in the AmpC algorithm are simple to perform, and easy to implement in the diagnostic laboratory.