Objective To determine the concentration of imipenem in human plasma by HPLC method, and establish the sampling process of clinical specimens based on stability investigation result. Methods The mobile phase was consisted of 0.01 mol·L^-1 ammonium acetate buffer(pH 6.8)-acetonitrile(95∶5, V∶V), and using Agilent Zorbax SB-AQ column(4.6 mm×250 mm,5 μm)to investigat the stability of imipenem under different temperature and with or without stabilizer. The injection volume was 30 μL, the colum temperature was 30℃, the flow rate was 1.0 mL·L^-1 and the detection wavelength was 298 nm. Results Imipenem had a good correlation coefficient(R²=0.999 1)at concentration of 3.30-105.60 μg·mL^-1. The lower limit of quantification was 0.41 μg·mL^-1, the intra-batch and inter-batch recovery rate were 97.83%-103.54% and 99.43%-104.24%, the intra-day and inter-day RSD were less than 15.0%. In stabilized plasma, imipenem could maintain stable at low temperature for 72 h and room temperature for 18 h, in non-stabilized plasma for 24 h and 6 h respectively. Conclusions This method was simple, rapid and accurate, which was suitable for the imipenem therapeutic drug monitoring, and establishing the sampling process of clinical specimen based on stability test could ensure the accuracy of plasma concentration monitoring.