BACKGROUND: This study establishes a UHPLC‒MS/MS method for the detection of zanubrutinib and explores its interaction with fluconazole and isavuconazole in rats. METHODS: A protein precipitation method using acetonitrile was used to prepare plasma samples using ibrutinib as an internal standard. Chromatographic separation and mass spectrometric detection of the analytes and internal standards were performed on a Shimadzu 8040 UHPLC‒MS/MS equipped with a Shim-pack velox C18 column (2.1 × 50 mm, 2.7 µm). Methanol and 0.1% formic acid-water were used as mobile phases. Intraday and interday precision and accuracy, extraction recoveries, and matrix effects of this method were determined. The linearity and sample stability of the method were assessed. Eighteen male Sprague‒Dawley (SD) rats were randomly divided into three groups with zanubrutinib (30 mg/kg) alone, zanubrutinib in combination with fluconazole (20 mg/kg) or zanubrutinib in combination with isavuconazole (20 mg/kg). Blood samples (200 µL) were collected at designated time points (ten evenly distributed time points within 12 h). The concentration of zanubrutinib was determined using the UHPLC‒MS/MS method developed in this study. RESULTS: The typical fragment ions were m/z 472.15 → 290.00 for zanubrutinib and m/z 441.20 → 138.10 for ibrutinib (IS). The range of the standard curve was 1-1000 ng/mL with a regressive coefficient (R2) of 0.999. The recoveries and matrix effects were 91.9-98.2% and 97.5-106.3%, respectively, at different concentration levels. The values for intra- and interday RSD% were lower than 9.8% and 5.8%, respectively. The RSD% value was less than 10.3%, and the RE% value was less than ± 4.0% under different storage conditions. Analysis of pharmacokinetic results suggested that coadministration with isavuconazole or fluconazole significantly increased the area under the curve (1081.67 ± 43.81 vs. 1267.55 ± 79.35 vs. 1721.61 ± 219.36), peak plasma concentration (332.00 ± 52.79 vs. 396.05 ± 37.19 vs. 494.51 ± 130.68), and time to peak (1.83 ± 0.41 vs. 2.00 ± 0.00 vs. 2.17 ± 0.41) compared to zanubrutinib alone. CONCLUSION: This study provides information to understand the metabolism of zanubrutinib with concurrent use with isavuconazole or fluconazole, and further clinical trials are needed to validate the results in animals.