Abstract A rapid and highly sensitive liquid chromatography-tandem mass spectrometric (LC–MS/MS) method for determination of doxofylline on rat dried blood spots and urine was developed and validated. The chromatographic separation was achieved on a reverse phase C18 column (250mm×4.6mm, 5μm), using 20mM ammonium acetate (pH adjusted to 3.5 with trifluoroacetic acid) and acetonitrile (75:25 v/v) as a mobile phase at 25°C. LC–MS detection was performed with selective ion monitoring using target ions at m/z 267 and m/z 195 for doxofylline and caffeine used as internal standard respectively. The calibration curve showed a good linearity in the concentration range of 1–5000ng/mL. The effect of hematocrit on extraction of doxofylline from DBS was evaluated. The mean recoveries of doxofylline from DBS and urine were 93.46 and 89.86% respectively. The intra and inter-day precisions were less than 4.28% in DBS as well as urine. The limit of detection and quantification were 0.24 and 0.84ng/mL in DBS and 0.28 and 1.00ng/mL in urine samples respectively. The method was validated as per ICH guidelines and successfully applied to a pharmacokinetic study of doxofylline in rats.