Late-night salivary cortisol (LNSC) is used as a screening test for Cushing's syndrome (CS), but there is no community derived normative data for the normal upper limit in the South Asian population. This study aimed to determine the upper limit of normal (97.5th centile) for late night salivary cortisol in an Asian Indian population using commercially available second generation ECLIA.LNSC in apparently healthy community dwelling subjects was assessed by multi stage cluster sampling. Healthy subjects 18 to 60 years of age from 8 Urban and 8 rural clusters of Thiruvananthapuram district were studied. Thirty subjects from an approximate population of 1000 persons from each cluster participated in the study. Saliva sample was collected between 2300hrs and 0000hrs and analysed using Roche COBAS-e-411 and ultrasensitive Cortisol-II kits the next day.Cortisol values from 474 salivary samples were available for final analysis after exclusion of improperly collected samples. The 97.5thcentile of the LNSC concentrations was 0.25mg/dl (6.89nmol/l) [90% Confidence Interval (CI) 0.23-0.27μg/dl i.e. 6.34-7.45 nmol/l]. In post-menopausal subjects, median LNSC was significantly higher but the 90% CI for upper limit of their LNSC (0.28mg/dl or7.72nmol/l) overlapped with that of premenopausal subjects.This study establishes the normal value of late-night salivary cortisol estimated by second generation ECLIA in healthy community dwelling Asian Indian subjects for the first time. Salivary cortisol at 2300-0000hrs is less than 0.25μg/dl(6.89nmol/l)in normal Asian Indian population. Menopause causes a significant increase in LNSC and may lead to over diagnosis of CS if not interpreted carefully.