An HPLC method was developed for assay and purity control of ursodeoxycholic acid, which is separated from all related compounds. Using an evaporative light scattering mass detector (ELSD), equipped with a micronebulizer and a narrow bore column, the bile acids (ursodeoxycholic acid, ursocholic acid, cholic acid, chenodeoxycholic acid, deoxycholic acid and litocholic acid) were efficiently separated on an Hypersil ODS-RP-18 column (100 × 2.1 mm) with methanol-acetonitrile-water (60 : 22: 18), adjusted at pH 4.00 with acetic acid, as mobile phase. Linearity response, accuracy, and precision of the ELSD over the range of sample amounts of interest were established for all compounds. The method demonstrates a suitability of the detector for the assay of ursodeoxycholic and related impurities and was applied for the analysis of ten pharmaceutical preparations.