Purpose: To develop an UPLC-MS/MS method for the quantitative analysis of pentoxifylline in beagle dog plasma and apply it to a pharmacokinetic study of food effect.Methods: Sample separation was achieved using a Kinetex Phenyl-Hexyl column (50 x 2.1 mm, 1.7 mu m) with a gradient elution program in 5.5 min after a simple protein precipitation with methanol. Using the mobile phase that made up by 0.2% formic acid and 5mM ammonium formate water (A) and methanol (B). Quantitation was carried out using the positive ionization mode with multiple reaction monitoring (MRM). A randomized, single-dose, two-period crossover study was conducted in six fasted or fed beagles that received 400 mg pentoxifylline sustained-release tablets (Brand name: Shuanling (TM), CSPC Pharmaceutical Group). WinNonlin (R) software was used to calculate pharmacokinetic parameters.Results: The linear calibration range was 2-1000 ng/mL (r(2)> 0.99). Both intra- and inter-batch precision were less than 6.27%, and the accuracy ranged from 88.65% to 97.18%. Pentoxifylline was readily absorbed in fasted and fed dogs administered a dose of 400 mg (t(max):1.54h vs 1.83h). Compared to the fasted group, the AUC(0 -> t) and C-max in the fed group increased by 1.71-fold and 1.30-fold, respectively. In the fasted group, the AUC(0 -> t) and C-max values were 4684.08 h center dot ng/mL and 2402.33 ng/mL, respectively. In the fed group, these values were 8027.75 h center dot ng/mL and 3119.67 ng/mL. The difference in AUC(0-t) between the fed and fasted group was statistically significant.Conclusion: The novel optimized UPLC-MS/MS assay is an effective tool for the determination of pentoxifylline and has been successfully applied in pharmacokinetic studies of pentoxifylline in beagle dogs. The administration of pentoxifylline sustained-release tablets with food significantly increased the area under the time curve, and it is recommended that they should be administered during or shortly after feeding.