A simple validated high-performance liquid chromatography (HPLC) assay was developed for determination of diflunisal and naproxen in human plasma samples. This is to compare the bioavailability of diflunisal-naproxen fixed-dose combination (FDC) with their separate dosage forms. The in vitro dissolution study was adopted to compare the dissolution behavior of FDC with respect to separate marketed tablets. In vivo study was conducted according to a single-center, randomized, single-dose, laboratory-blinded, 2 Way, Cross-Over Study with a washout period of 10 days. Under fasting conditions, 24 healthy Egyptian male volunteers were randomly allocated to receive a single oral dose of either one FDC tablet or co-administration of two separate diflunisal and naproxen marketed tablets. Plasma samples were obtained over a 72-h interval and analyzed for diflunisal and naproxen by reversed phase liquid chromatography with UV detection. The pharmacokinetic parameters Cmax, AUC0-t, AUC0-∞, tmax, and t1/2 were determined from plasma concentration-time profiles. The 90% confidence intervals for the ratio of log transformed values of Cmax, AUC0-t, and AUCt-∞ of the 2 treatments were within the acceptable range (0.8-1.25) for bioequivalence. From pharmacokinetic and in vitro studies perspectives, 1 FDC tablet demonstrated similar relative bioavailability with the 2 individual -reference tablets.