A simple and reliable HPLC method was developed and validated for determination of rofecoxib in bovine serum albumin microsphere. The analyses were performed on a C18 column (150 x 4.6 mm, 5 mu m particle size) at room temperature with UV detection at 272 nm. The mobile phase was composed of acetonitrile-0.1% o-phosphoric acid solution in water (1:1, v/v) mixture, and flow rate was set to 1 mL/min. The method was validated according to the international guidelines with respect to stability, linearity range, limit of quantitation and detection, precision, accuracy, specificity, and robustness. The detection and quantification limit of the method were 1.0 mu g/mL and 2.5 mu g/mL, respectively. The method was linear in the range of 2.5-25 mu g/mL with excellent determination coefficients (R-2 >0.99). Intra-day and inter-day precision (<1.76% RSD) and accuracy (<0.55 % Bias) values of the method also fulfilled the required limits. It was concluded that the developed method was accurate, sensitive, precise, and reproducible according to the evaluation of the validation parameters. The applicability of the method was confirmed for in vitro quantification of rofecoxib in bovine serum albumin microspheres.