OBJECTIVE：To establish a method for the content determination of zolpidem tartrate in microsamples of rat plasma. METHODS：Rats were given zolpidem tartrate solution 3 mg/kg intragastrically，and 0.2 ml blood sample were collected and isolated. 50 μl plasma was precipitated by methanol，and the supernatant was determined by HPLC-fluorescence combined with external method. Agilent HC-C18 column was used with mobile phase consisted of 0.03 mol/L KH2PO4 solution（containing 0.2％ triethylamine）- methanol（33 ∶ 67，V/V）at flow rate of 1.0 ml/min. The excitation and emission wavelengths were 254 nm and 390 nm，respectively. The sample size was 20 μ l. RESULTS：The linear ranges of zolpidem tartrate in plasma was 2-200 μg/L（r＝ 0.999 7），and the limit of quantification was 2 μg/L. The method recoveries of zolpidem were（96.96±1.35）％-（105.0±5.36）％ （RSD＝2.20％-4.88％，n＝5），and extraction recoveries were（79.72±0.01）％-（80.77±0.02）％（RSD＝1.34％-3.90％，n＝5）. The intra-day and inter-day RSDs were 1.40％-5.10％ and 3.22％-9.25％（n＝5），respectively. CONCLUSIONS：The method is simple，sensitive and suitable for the content determination of zolpidem tartrate in microsamples of plasma.