BACKGROUND AmpC β-lactamases are Ambler class C enzymes that confer resistance to extended spectrum cephalosporins and are not inhibited by β-lactamase inhibitors. These enzyme-producing organisms produce infections that are associated with significant morbidity and mortality. Resistance to third-generation cephalosporins develop in these organisms after exposure to these agents. This complicates treatment options and carbapenems are considered optimal. MATERIALS AND METHODS In this cross-sectional study, AmpC β-lactamase production was determined in Gram-negative clinical isolates from various clinical samples. Isolates resistant to cefoxitin and third generation cephalosporin (3GC) antibiotics were tested for the production of AmpC β-lactamases by using an inhibitor-based method (IBM) with phenylboronic acid. RESULTS It was observed that, among the 100 Gram-negative isolates, 48 (48%) were resistant to cefoxitin. Using IBM, the occurrence of AmpC β-lactamases was found in 24 (24%) of these 48 isolates. ESBL/AmpC co-carriage was found in 13 (13%) of these isolates by E-Test. Among the 24 AmpC positive isolates, 10 (41.6%) were E. coli, 5 (20.8%) were Enterobacter cloacae, 5 (20.8%) were Klebsiella pneumoniae, 2 (8.3%) were Acinetobacter baumannii and 2 (8.3%) were Pseudomonas aeruginosa. CONCLUSION AmpC production can be determined in routine clinical microbiology laboratory using IBM as it is a simple, rapid and technically easy procedure. Thus, their accurate detection and characterisation plays an important role in their epidemiological survey, infection control and treatment.