Down syndrome (DS) has major resource implications especially in developing countries being third most important cause of mental handicap. Maternal serum screening for chromosomal aneuploidies and neural tube defects (NTDs) is practiced worldwide in many countries and has been integrated into mainstream health care, while it is gradually gaining momentum in Asian countries. This prospective cohort study was carried out in pregnant women undergoing triple screening test between January 2007 and December 2010 after informed consent. Biomarkers alpha-fetoprotein, human-chorionic-gonadotropin and unconjugated-estriol were tested, and risk of pregnancy being affected with DS, Edward's syndrome or NTDs were calculated. Screen-positive patients were referred for detailed ultrasonography and confirmatory amniocentesis. Follow-up record was maintained until delivery. Of 7400 pregnant women enrolled, 419(5.7%) were screen-positive, including 339 positive for DS, two for trisomy 18, and 62 for NTDs. Total eight cases of DS were eventually diagnosed in the population (prevalence of DS = 1 : 925), seven of which were detected in utero following diagnostic evaluation for positive serum screen (DR of DS screen = 87.5%). Total five cases of NTD were observed, yielding NTD prevalence of 0.67/1000. Triple screening in the second trimester is reasonably effective for the detection of major chromosomal defects and NTDs, and can be implemented successfully also in India.