Background:Multidrug resistance is spreading rapidly among bacterial species around the world. The production of beta-lactamases is responsible for the development of antibiotic resistance. Detecting these resistance mechanisms presents a significant challenge to clinical laboratory. Carbapenemase is a metallo -β-lactamase enzyme that can develop drug resistance. The current investigation sought to detect the synthesis of ESBL (Extended Spectrum Beta Lactamases), Amp C and carbapenemase in Enterobacteriaceae isolated from a tertiary care centre.Objectives:The study aims to analyse the antibiotic susceptibility of Enterobacteriaceae and compare phenotypic approaches to a 12-disk procedure for detecting co-expression of β-lactamases.Methods:A cross sectional study conducted with 200 isolates of Enterobacteriaceae members. ESBL, Amp C and MBL production was detected by Phenotypic methods along with 12 disk method.Result:Out of 200 samples, 122 (61%) were from adults and male to female ratio was 1.17:1. Most common species isolated was Klebsiellapneumoniae (46.7%). Most of the isolates were resistant to routine antibiotics used. Of the total, 72% were ESBL & 27.5% were AmpC producers. MHT and mCIM identified 43% and 60.5% of carbapenemase producers respectively. 12 disk method identified 21%, 9.5% and 24% of ESBL, AmpC&carbapenem producers respectively. 3.5% of ESBL+AmpC producers, 26% of ESBL &carbapenemase producers, 4.5% of AmpC and carbapenemase producers. 6% isolates were ESBL+AmpC+ Carbapenemase producers.Conclusion:Production of beta-lactamases mediates antibiotic resistance in clinical isolates. The development of simple and inexpensive screening methods to detect beta-lactamase production in laboratories is crucial for optimal treatment of critically ill and hospitalized patients, and to control the spread of resistance.