An analytical method based on Liquid-liquid extraction has been developed and validated for analysis of Tadalafil in rat plasma. Tadalafil-D3 was used as an internal standard. Zorbax-SB C18, 75 x 4.6 mm, 3.5 μm column provided chromatographic separation of analyte followed by detection with mass spectrometry. The method involves simple isocratic chromatographic condition and mass spectrometric detection in the positive ionization mode using an API-4200 system. The total run time was 3.0 minutes. The proposed method has been validated with linear range of 0.50–1000.00 ng/mL for Tadalafil. The intra-run and inter-run precision values are within 1.37 - 2.25% and 2.23 - 5.31%. The overall recovery for Tadalafil and Tadalafil-D3 was 91.07% and 86.66%. This validated method was applied successfully in rat plasma samples for pharmacokinetic study
