Aim: Hormonal milieu of pregnancy may be an important determinant of outcome in both mother __ampersandsignamp; fetus. Estimation of trimester specific hormone concentrations measured may serve as a yard stick to predict feto-maternal outcome. However, the literature review suggest that the reference interval of progesterone established in western pregnant population may not be applicable in India with different reproductive profile. The primary objective of this study was to estimate trimester specific and assay-specific reference interval of progesterone in healthy pregnant females. Methodology: About 276 healthy pregnant ladies were enrolled as study participants with a distribution of 81, 111, and 84 in 1st, 2nd __ampersandsignamp; 3rd trimester respectively. The study was conducted after obtaining ethical approval of Nil Ratan Sircar Medical College __ampersandsignamp; subjects were collected using pre-defined inclusion and exclusion criteria. Serum of study subjects was analysed for progesterone by Enzyme linked immunesorbent assay. Results: The serum progesterone level was found to increase gradually throughout pregnancy. Conclusion: The study helped us to determine the reference interval in healthy pregnant female and the reference values markedly differ from the established reference interval in other population.