Great attentions have been drawn by quinoline for its broad bioactivity as anti-fungal, anti-bacterial and anti-tumor activities. Compared with cisplatin, 83b1, a quinoline derivative, showed equal activity in anti-tumor and lower cyctotoxicity in normal cell. In this study, a simple, rapid and sensitive method for determination of 83b1 in rat plasma using UHPLC–MS/MS was developed for the first time. Loratadine was used as an internal standard (IS). Separation was performed on an Xterra MS C18 column by isocratic elution using acetonitrile: water solution with 1‰ formic acid (90:10, v/v) as mobile phase at a flow rate of 0.3 mL/min. A triple quadrupole mass spectrometer operating in the positive ion-switching electron spray ionization mode with selection reaction monitoring (SRM) was employed to determine 83b1 and IS transitions of m/z 321.82 → 147.84, 382.71 → 258.76 for 83b1 and Loratadine, respectively. The values of specificity, linearity and lower limit of quantification, intra- and inter- day precision and accuracy, extraction recovery, matrix effect and stability for this method satisfied the acceptable limits. The lower limit of quantification was 0.5 ng/mL with a linear range of 0.5–1500 ng/mL. The validated method was employed to study the bioavailability of 83b1 in rat by dosing with intravenous injection (1 mg/kg) and gavage (10 mg/kg), and the oral bioavailability of 83b1 in rat was calculated as 20.9 ± 8.8%.