OBJECTIVE To establish an ultra-high-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for the determination of doxorubicin (DOX) and its active metabolite doxorubicinol (DOXol) in mouse plasma and heart, liver, spleen, lung, kidney and tumor tissues, and to explore the pharmacokinetic effects of paclitaxel (PTX) on DOX and DOXol. METHODS The daunorubicin (DAU) was used as the internal standard. The mobile phase consisted of acetonitrile (A)-0.1% formic acid water (B) for gradient elution:0 min 10% A, 0.5 min 30% A, 1.0 min 60% A, 1.5 min 60% A, 2.0 min 10% A, 2.2 min 10% A, and the separation was carried out on an ACQUTTY UPLC BEH C₁₈ column(2.1 mm×50 mm, 1.7 μm). The transitions of m/z 544.25→396.96, m/z 546.25→399.00, m/z 528.38→321.00 were used to quantify DOX, DOXol, and DAU. UPLC-MS/MS method was used to determine the concentration of DOX and DOXol in plasma and tissues of Balb/C mice at different times in the three groups. The pharmacokinetic differences of DOX and DOXol were compared between the single administration group and the combined administration group. RESULTS DOX and DOXoL in the plasma and tissues of mice showed a good linear relationship (r>0.99), the intra-and inter-day precision (RSD) was <6.9%, the accuracy (RE) was ±6.2%, the extraction recovery rate was 93.1%-107.4% and the matrix effect was 85%-115%. The detection method was stable and reliable. The concentration of DOX and DOXol in each tissue was kidney>liver>spleen>lung>heart>tumor, and DOXol was not detected because its concentration in the tumor was lower than the lower limit of quantitation. Compared with the DOX alone group, the concentrations of DOX and DOXoL in the plasma, liver and kidney were increased within 4 h after administration in the PTX→DOX group. CONCLUSION The method is highly specific and sensitive, and can be used for pharmacokinetic studies of DOX and DOXol. PTX affects the distribution of DOX in mice, but has no significant effect on the cardiac distribution of DOX and DOXol.