A simple, rapid and reliable high performance liquid chromatography method (HPLC) with ultraviolet detection (UV) was developed and validatedaccording to ICH guidelines, for quantitative analysis and therapeutic drug monitoring of diclofenac sodium (DS) in human plasma. Plasma samples (0.7mL) were acid hydrolysis by 100 µL, 1 M hydrochloric acid. Analytes were concentrated from plasma by liquid–liquid extraction with 2 mL ethyl acetateby repeated twice, which allows to obtain good extraction yields (98.75%-99.32%). The separation was achieved by employing C18 analytical column(3.5 µm particle size, 150 mmx3.9 mm I.D.) under isocratic conditions using acetonitrile and NaH2PO4 mixture (42.5:57.5, v/v) as mobile phase (pH:3.16) flow rate of 1.5 mL/min. Naproxen (3 µg/mL) was used as an internal standard (IS). The DS and IS were detected at 281 nm and eluted at 2.6 and6.2 min, respectively. Total run time was 7 min. Method showed linearity with very good determination coefficients (r2=0.999), over the concentrationrange of 50 - 1600 ng/mL. Limits of detection (LOD) and quantification (LOQ) were 8.95 ng/mL and 27.12 ng/mL, respectively. Intra-day precisionand accuracy were between 0.93-5.27; 1.74-9.81, respectively. Inter-day precision and accuracy were between 2.71-6.64; 2.03-9.16, respectively. Thismethod was successfully applied for determination of DS plasma concentrations during a pharmacokinetic study in healthy volunteers (n=12) after anoral administration of Voltaren® 75 mg/tablet and remarkable variations in DS levels were observed. In our study, on the contrary to equivalent dosesof DS, the observed significant differences in plasma levels of DS, on 2nd, 4th and 6th hours, can be explained by pharmacokinetic differences, that arisefrom mainly polymorphisms of CYP2C9 and CYP3A4, which are major enzymes responsible for DS metabolism.