The first-order multivariate calibration method was used for the simultaneous determination of the camptothecin derivative, CPT-11 and its main metabolite, SN-38. The method is based on the fluorescence emission of these compounds in acidic media (acetate buffer, pH 4.75) and in the presence of ethanol (50%). The experimental calibration matrix was constructed with 12 samples using a central composite design. The cross-validation method was used for selecting the optimum number of factors. The results showed that simultaneous determination could be performed in the range 0.08 - 1.50 and 0.10 - 0.40 µg mL(-1) for CPT-11 and SN-38, respectively. The method was successfully applied to the simultaneous determination of both analytes in human urine and in serum samples previously extracted with chloroform.