The objective of this study was to investigate the myotoxicity potential of the solvents used in the preparation of polymer solutions and O/W-in situ forming microparticle (ISM) systems. The acute myotoxicity studies of the tested solvents, emulsions of the solvents, polymer solutions as well as the O/W-ISM formulations with varying phase ratios were investigated using the in vitro isolated rodent skeletal muscle model by measuring the cumulative creatine kinase (CK) efflux. Phenytoin and isotonic sodium chloride solution served as positive and negative controls, respectively. Results from the in vitro myotoxicity studies suggested that the investigated five partially water miscible solvents caused muscle damage in the following rank order: benzyl alcohol>triethyl citrate>triacetin>propylene carbonate>ethyl acetate. Myotoxicity of ethyl acetate was found to be comparable to that of the isotonic sodium chloride solution. Emulsions of the undiluted solvents and an aqueous 0.5% Pluronic F 68 solution (ratio 1:4) could dramatically reduce the myotoxicities to 24-65%. The myotoxicity of O/W-ISM was less than those of the polymer solutions and the undiluted solvents. The cumulative CK level from the muscle treated with the O/W-ISM with phase ratio 1:4 was comparable to those from the negative controls. Area under the CK plasma curve from Sprague-Dawley rats was used to evaluate the in vivo myotoxicity following an intramuscular injection of the formulations. The in vivo myotoxicity data was well correlated with the in vitro myotoxicity data and confirmed the good muscle compatibility of the O/W-ISM formulations.