OBJECTIVE： To develop a method for simultaneous determination of irinotecan （CPT-11） and its active metabolite 7-ethyl-10-hydroxycamptothecin （SN-38） in human plasma. METHODS： After precipitated by acetonitrile and acidified with hydrochloric acid， using camptothecin as internal standard， the plasma sample was determined by HPLC-FLD. The determination was performed on Waters Luna C18 column with mobile phase consisted of 0.05 mol/L sodium dihydrogen phosphate-acetonitrile （70 ∶ 30，V/V， adjusted pH to 4.0 by phosphoric acid） at flow rate of 1 mL/min. The excitation wavelength was set at 380 nm； the emission wavelengths of CPT-11 and SN-38 were set at 480 nm and 535 nm， respectively. The column temperature was 25 ℃ and the sample size was 20 μL. RESULTS： The linear ranges were 200-1 000 ng/mL for CPT-11 （r＝0.999 4，n＝5） and 5-45 ng/mL for SN-38 （r＝0.999 2，n＝5）. RSDs of inter-day and intra-day were 1.68％-5.57％. The relative recoveries of CPT-11 and SN-38 were 90.12％-106.93％（RSD＜8％，n＝5） and 92.07％-102.56％（RSD＜6％，n＝5）； the extraction recoveries of CPT-11 and SN-38 were 72.23％-86.56％（RSD＜6％，n＝5） and 71.98％-83.44％（RSD＜7％，n＝5），respectively. The plasma concentrations of CPT-11 and SN-38 in 5 patients with colon cancer were 431.13-617.19， 13.97-31.89 ng/mL（1 h after intravenous dripping） and 398.14-584.43， 11.61-29.94 ng/mL（2 h after intravenous dripping）. CONCLUSIONS： The method is simple， rapid， sensitive， reproducible and suitable for the determination of plasma concentration and pharmacokinetic study of CPT-11 and its metabolite SN-38.