AmpC beta-lactamases (Bla(Ampc)) are an emerging group of antimicrobial resistance determinants. The lack of an agreed Bla(Ampc) detection method hinders investigation of their epidemiology and understanding of their clinical significance. This study compared the sensitivity and specificity of phenotypic methods of Bla(Ampc) detection in a collection of 246 Enterobacteriaceae with a diverse range of beta-lactam resistance profiles. The Bla(Ampc) screening methods evaluated were based on cephamycin, ceftazidime and cefepime susceptibility. These were compared with Bla(Ampc) screening using conventional ESBL detection methods. The confirmatory methods evaluated were biologically based assays, inhibitor-based assays, an AmpC Etest and a rapid chromogenic assay. A multiplex nucleic acid amplification test and the three-dimensional enzyme extraction assay were used as reference methods. Bla(Ampc) activity was present in 74 isolates. The majority of the enzymes were plasmid-encoded and belonged to the CMY, DHA and EBC families. The screening methods had sensitivities between 47 and 99% and specificities of 45-95%. The performance of confirmatory tests varied widely, ranging in sensitivity from 19% to 97% and in specificity from 88% to 100%. Only the Tris-EDTA and MAST ID D68C disc tests had a sensitivity and a specificity above 90%. Further investigation is needed to establish the most suitable enzyme substrates, inhibitor types, inhibitor concentrations and interpretative cut-offs in order to refine the inhibitor-based methods. A simple disc-based protocol using cefoxitin non-susceptibility as a screening tool, followed by the Tris-EDTA method for confirmation, detects Bla(Ampc) activity with 95% sensitivity and 98% specificity.