Objective To establish a method for measuring serum content of gentiopicroside in Beagle dogs and to explore its internal pharmacokinetic features. Methods The rapid and sensitive HPLC method was adopted as external standard. After intravenous injection of gentiopicroside, serum samples of the Beagle were collected and measured on C18 reverse-phase chromatographic column after liquid -liquid extraction. Results The linear range of gentiopicroside was 0.1343～687.375 ?g?mL-1(r2=0.99993). The recovery was within 81.15 %～97.99 %. The limit of detection (LOD) was 0.1343 ?g?mL-1. Conclusion The pharmacokinetic process of gentiopicroside can be described as one-compartment model with intravenous injection , t1/2 = 1.1324 h, Ke = 0.5811 h-1, V= 0.5316 L?kg-1, CL = 0.3274 L?kg-1?h-1. This indicates that gentiopicroside can be quickly distributed and eliminated and is not liable to be accumulated in the body.