Abstract: OBJECTIVE To establish a method for determination of pseudoephedrine in urine samples of healthy volunteers. METHODS The pseudoephedrine was determined by LC-MS/MS. The separation was carried on an Agilent Eclipse plus C₁₈(4.6 mm×100 mm, 3.5 μm) colume with a mobile phase of methanol-0.005 mol?L^-1 ammonium formate apueous solution with 0.1% formic acid (3∶1) at the flow rate of 0.5 mL?min^-1, The column formate temperature was set at 40 ℃. ESI source was applied and operated in positive ion mode. Quantitative determination was performed by using selective reaction monitoring (SRM) of m/z 166.2→148.1 for pseudoephedrine and m/z 152.1→110.1 for paracetamol used as internal standard. RESULTS Pseudoephedrine in urine samples showed good linearity in the range of 500?300 000 μg?L^-1(r²=0.998). The intra-batch and inter-batch precisions(RSD) were ≤4.41% and ≤10.04%, respectively. The extraction recoveries of pseudoe- phedrine and internal standard were more than 90%. The average cumulative excretion of pseudoephedrine in urine was 44.57%. CONCLUSION The method that using LC-MS/MS to determine pseudoephedrine in urine is accurate, simple and sentitive, and it has a good reproducibility. This method can be applied to the clinical pharmacokinetic study and therapeutic drug monitoring of pseudoephedrine.