In this study, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated to simultaneously determine the anticancer drugs etoposide and paclitaxel in mouse plasma and tissues including liver, kidney, lung, heart, spleen and brain. The analytes were extracted from the matrices of interest by liquid-liquid extraction using methyl tert-butyl ether-dichloromethane (1:1, v/v). Chromatographic separation was achieved on an Ultimate XB-C-18 column (100 x 2.1mm, 3m) at 40 degrees C and the total run time was 4min under a gradient elution. Ionization was conducted using electrospray ionization in the positive mode. Stable isotope etoposide-d3 and docetaxel were used as the internal standards. The lower limit of quantitation (LLOQ) of etoposide was 1ng/g tissue for all tissues and 0.5ng/mL for plasma. The LLOQ of paclitaxel was 0.4ng/g tissue and 0.2ng/mL for all tissues and plasma, respectively. The coefficients of correlation for all of the analytes in the tissues and plasma were >0.99. Both intra- and inter-day accuracy and precision were satisfactory. This method was successfully applied to measure plasma and tissue drug concentrations in mice treated with etoposide and paclitaxel-loaded self-microemulsifying drug-delivery systems.