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 DCM: MTBE (1:1, v/v). Chromatographic separation was achieved on an Ultimate XB-C18 column (100 mm × 2.1 mm, 3 μm) at 40 °C and the total run time was 4 min under a gradient elution. Ionization was conducted using electrospray in the positive mode. Stable isotope etoposide-d3 and docetaxel were used as the internal standards (IS). The lower limit of quantitation (LLOQ) of etoposide was 1 ng/g tissue for all tissues and 0.5 ng/mL for plasma. The LLOQ of paclitaxel were 0.4 ng/g tissue and 0.2 ng/mL for all tissues and plasma, respectively. The coefficients of correlation (R2 ) for all the analytes in the tissues and plasma were greater than 0.99. Both intra-day and inter-day accuracy and precision were satisfactory. This method was successfully applied to measure plasma and tissues drug concentrations in mice treated with etoposide and paclitaxel-loaded self-microemulsifying drug delivery systems.