A procedure for the simultaneous kinetic spectrophotometric determination of cephalexin and trimethoprim was described. It was based on the different reaction rate of oxidation of these compounds with yellow ammonium cerous (Ⅳ) sulfate in acidic medium and colorless cerous (Ⅲ) sulfate was produced. The overlapped kinetic data was quantitatively resolved by the use of chemometric methods, partial least squares (PLS), principal component regression (PCR) and radial basis function-artificial neural network (RBF-ANN). The proposed method was also applied to the simultaneous determination of cephalexin and trimethoprim in pharmaceutical preparation and human urine with satisfied results, which compared well with those obtained by HPLC.