Abstract Purpose Bile acids are a group of cholesterol metabolites functioning as key regulators of glucose, lipid, and energy metabolism. Their homeostatic control is essential to the physiology of the normal pregnancy. Abnormalities of bile acids regulation in pregnancy lead to intrahepatic cholestasis of pregnancy, a serious condition associated with a number of fetal and maternal morbidities. Dysregulation of glucose and lipids is also tied to perturbations in bile acid concentrations. Changes in bile acid metabolic profiles in the second and third trimesters of pregnancy have been incompletely explored. We seek to establish pregnancy-specific normative ranges for a number of bile acids in women in the second and third trimesters and explore changes in their concentrations in the period from 12 to 40 weeks gestation. Procedure In this cross-sectional study, a total of 782 normal pregnant women were enrolled including n = 290 in the second trimester (12–28 weeks) and n = 492 in the third trimester (29–40 weeks). The concentrations of 14 bile acids were measured by liquid chromatography and mass spectrometry (LC-MS) and compared at various time points. Reference intervals of these bile acids were calculated using standard statistical techniques. Results A reference interval profile of 14 bile acids from a cohort of 782 normal pregnant women was developed. Significant differences in concentration were found between the second trimester and the third trimester. Unconjugated bile acids dominate the bile acid profile in the second trimester, while conjugated bile acids, especially (taurine-conjugated) dominate in the third trimester. 28–31 weeks gestation was the notable change period of bile acid metabolism. Conclusion This study establishes pregnancy-specific reference intervals for bile acids in the second and third trimesters. As bile acid composition changes with gestational age, this study establishes a foundation for trimester-specific clinical interpretation of bile acid metabolic profiles in pregnant women.