Comparison of the patient's laboratory results with reference intervals is one of the most fundamental tools for interpreting laboratory results, making a clinical decision and monitoring the course of the disease. The aim of this study was to introduce a method for determining bile acids into the analytical system, to verify its parameters, and to establish a reference interval for bile acids for the population of pregnant women from the 28th to the 42nd week of pregnancy. Bile acids as a specific biomarker are used to diagnose intrahepatic cholestasis of pregnancy, a disease that usually occurs in the third trimester and manifests itself as pruritus while being potentially fatal for the child. Verification of the method showed that bile acids are stable in serum samples for 2 days at 20 ℃, 14 days at 4-8 ℃, and 22 days at -20 ℃. The precision study met the criteria. Within-laboratory precision was 2.2% and 1.7% for the concentration of bile acids within the reference range and above the upper limit, respectively. Linearity was verified for the concentration range of 3.5-172.1 μmol/l, and the verification of the reference interval for adults met the criteria. The lipemia interference test showed a positive bias (%) of bile acid concentration with an increase in serum triglyceride concentration. Lipemia causes unreliable results at a SMOFlipid® 20% emulsion concentration of 10 g/L, i.e. at a triglycerides concentration above 12.4 mmol/L in samples with a low initial concentration of bile acids. Reference interval for bile acids was determined a priori. Total bile acids concentration was determined in 116 serum samples of the reference pregnant women who were selected based on the previously set exclusion and inclusion criteria. Statistical analysis of the results was carried out according to CLSI guidelines. The reference interval of 1.0 to 7.3 μmol/L for the population of pregnant women from the 28th to the 42nd week of pregnancy was determined using the non-parametric method.