The present study was conducted to isolation of Extended Spectrum Beta Lactamase (ESBL) producing in gram`s negative bacteria specially in Eschericia coli and Klebsiella pneumonia in different clinical samples received at the department of Laboratory medicine, Alchemist hospital, Gurgaon, india. Aerobic culture was performed in automated analyzer vitek-2 and isolate from blood agar and mackonky agar media. Antimicrobial susceptibility testing was carried out using the Kirby-Bauer agar diffusion method and ESBL production was detected on Mueller Hinton agar by double-disk synergy technique and vitek 2. A total of 180 isolates were collected from 493 samples. From these 180 samples two types of organisms were isolated: Escherichia coli and klebsiella sp. In some samples these two organisms become resistant to many ß lactam antibiotics like cefazoline, ceftriaxone, Aztreonam, Ceftazidime, Ampicillin etc. drug sensitivity test done by disc diffusion method as well as automated vitek-2. Percentage of ESBL positive E.coli was 70.5 % and Klebsiella sp. was 86.1 % and percentage of ESBL negative E.coli was 29.5 % and ESBL negative Klebsiella sp. was 14.1 %. However, theses finding shows prevalence of ESBL positive is more in Klebsiella species and negative is more in Escherichia coli on our hospital.