The study concerns development of simulated synovial fluid for in vitro release testing of drug and prodrug-based depot delivery systems for joint injection. The importance of drug protein binding and esterase-catalyzed prodrug hydrolysis was investigated using a membrane-based in vitro release testing system, the rotating dialysis cell. Protein binding was investigated in synovial fluid from arthritic patients, human plasma and phosphate buffer, pH 7.4, containing serum albumin. Compounds with low (sodium diatrizoate) and high (naproxen and diclofenac) degrees of protein binding were investigated. The cleavage of two poorly soluble naproxen and diclofenac ester prodrugs was studied in esterase-containing media, diluted synovial fluid and plasma. Regarding protein binding, the in vitro release studies revealed that drug release profiles obtained using buffer comprising 2% (w/v) serum albumin, or 40% (v/v) plasma were comparable to profiles obtained employing 80% (v/v) synovial fluid. For prodrug-based depot injectables, application of 40% (v/v) plasma provided release profiles comparable to those using 80% (v/v) synovial fluid. Design of simulated synovial fluids for characterizing in vitro the interplay between drug/prodrug solubility, protein binding and susceptibility to enzymatic cleavage, and joint residence time is feasible. Synovial fluid may be replaced with readily accessible serum albumin solutions or diluted plasma.