Abstract: Influences of conditions of chromatographic separation (i.e., strong elution, weak elution and gradient elution) on the matrix effect in the LC-MS/MS analysis with quantification by external standard method were studied by using S-phenylmercapturic acid as the target compound. It was shown that: ① in analyzing standard solutions of different concentrations without matrix separation by strong elution gave highest sensitivity and high frequency of sample introduction per unit time; ② in analyzing standard solutions of different concentrations with same matrix, significant improvement of the matrix effect was obtained by the weak elution, however, better result was achieved by the gradient elution; ③ in analyzing standard solutions of different concentrations with matrixes from different sources, the separation by gradient elution gave the best results, the efficiency of separation was raised with lowering of co-elution and the matrix effect on the analysis of the target compound was lowered effectively. Hence, the improving of conditions of chromatographic separation in LC-MS/MS analysis was of utmost importance for lowering the matrix effect.