Extraction and determination of warfarin were performed using supramolecular solvent-based microextraction (SSME) and high performance liquid chromatography (HPLC). Effective factors on SSME including pH and temperature of sample, stirring speed, volume of the solvent, salt effect, and extraction time were investigated and optimized. Determination of warfarin was performed by HPLC using a mobile phase consisting of methanol and (0.5 %) phosphoric acid solution (80:20 v/v) and UV detector set at 320 nm. Under the optimized conditions pH of sample solution = 3, sample temperature = 30 °C, stirring speed = 800 rpm, volume of the solvent = 30 µL, obtained. The limit of detection was in the range of 0.4–14.5 ng mL−1. Linearity of the method was determined in the range of 14.5–300.0 ng mL−1for plasma and 2.9–300.0 ng mL−1for urine with coefficient of determination (R2) ranging from 0.9975 to 0.9985. The method was applied successfully for determination of warfarin in plasma and urine samples.
