There are currently no standardized phenotypic methods for the screening and detection of AmpC enzymes. This study aimed to evaluate different methods to detect AmpC enzymes in Escherichia coli, Klebsiella spp., and Proteus spp., comparing the results from two disk-based methods and an agar dilution method. AmpC activity was determined for 255 clinical isolates by use of a three-dimensional enzyme assay combined with a multiplex PCR assay for plasmid-borne ampC genes. These results were compared against a disk-based inhibitor assay using various combinations of cefpodoxime and cefoxitin as antibiotic substrates and boronic acid or cloxacillin as an AmpC inhibitor. The presence of enzyme induction by disk approximation was evaluated using imipenem, cefoxitin, and amoxicillin-clavulanate as inducing agents against ceftazidime. Finally, an agar dilution assay was performed, using cefoxitin with and without added cloxacillin. AmpC activity was present in 49.8% of test isolates, 93.7% of which were positive for plasmid-borne ampC genes. CIT-like enzymes were predominant in E. coli, and DHA-like enzymes were predominant in Klebsiella spp. The disk-based inhibitor tests performed better than the agar dilution assay, while detection of AmpC by disk induction had a poor sensitivity. The cefoxitin-cloxacillin disk combination provided the best overall performance, with a sensitivity and specificity of 95%. This study confirmed the accuracy of disk-based inhibitor screening for AmpC enzymes, which proved reliable at detecting CIT- and DHA-like plasmid-borne ampC genes. The methods are simple enough for introduction into clinical microbiology laboratories.