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Abstract: A reverse phase ultra-performance liquid chromatography coupled with evaporative light scattering detection (UPLC–ELSD) method was successfully established for simultaneous determination of five bile acid derivatives in Calculus Bovis and its medicinal preparations using a Waters Acquity UPLC T3 C18 column (2.1×50mm, 1.8μm) with gradient elution of 0.2% aqueous formic acid and acetonitrile. The influence of liquid flow-rate and column temperature, gas flow-rate and drift tube temperature on resolution was investigated to get the optimized parameters and conditions. This method was validated according to the regulatory guidelines with respect to precision, accuracy and linearity. Under optimum conditions, the five analytes were baseline separated in 11min with detection and quantification limits ranged in 3.0–6.0ng and 6.0–18.0ng, respectively, which provided about a twofold reduction in analysis time and good efficiency and sensitivity by comparing a conventional high performance liquid chromatography (HPLC) method. [Copyright &y& Elsevier]