An attempt was made to speciate and antimicrobialy differentiate 150 clinically significant isolates of CoNS by practical scheme adapted from various references. Total 150 isolates were collected from NICU, Pediatrics Department, CSMMU, Lucknow from different sites and subjected to antimicrobial screening and biochemical characteriza-tion using conventional microbiological methods. Antibiotic sensitivity test was performed for all clinically significant isolates. 90% isolates were conveniently identified. S.epidermidis (40%), S.saprophyticus (14%), S.haemolyticus (12%), S.hominis (6%), S.lugdunensis (6%), S.schleiferi (2%), S.capitis (4%), S.warneri (6%), 15 isolates were not identified to the species level. Antibiotic susceptibility testing showed maximum resistance to ampicillin and penicillin with 80% and 38% strains showed the resistance to oxacillin. The increasing recognition of pathogen potential CoNS and emergence of drug resistance among them demonstrates the need to adopt simple laboratory procedure to identify and de-termine the prevalence and antibiotic sensitivity of CoNS. It will help in treating clinicians for first line treatment in hospital.