Background and objectives: Gestation is associated with profound hormonal and metabolic changes in the mother. These alterations facilitate the placenta to take over the dominant role of steroid production. The present study was designed to assess the concentrations of estrogen, progesterone, and total testosterone in normal pregnancy. Materials and methods: Fifty normotensive normal pregnant subjects with mean age 26.4 ± 4.48 years with no history of hypertension, vomiting, fever, cough, and cold were taken. Their mean ± SD gestational age at the time of study was 23.8 ± 10.2 weeks, who attended to the gynecology OPD were included in the study. Age-matched 50 nonpregnant subjects, not having any acute illness, thyroid, liver, and renal diseases, were taken as control. Serum estrogen, progesterone, and total testosterone were estimated by chemiluminescent method on Immulite 1000. Results: The alterations of serum reproductive hormone levels in normotensive pregnant subjects were found when compared to those of non-pregnant control group. Study group showed a significant (p value < 0.001) increase in serum estrogen, progesterone, and total testosterone levels due to production of prostaglandin before labor, subsequent conversion of cholesterol to progesterone in the placenta, and defect in newborn growth and size, respectively. When compared to nonpregnant control group. Conclusion: Reproductive hormones such as estrogen, progesterone, and total testosterone in normal pregnancy are of paramount importance during pregnancy. This study suggests that levels of abovementioned parameters were altering in normal physiological changes during pregnancy.