Identification of factors that improve muscle function in boys with Duchenne muscular dystrophy (DMD) could lead to an improved quality of life. To establish a functional in vitro assay for muscle strength, mdx murine myoblasts, the genetic homologue of DMD, were tissue engineered in 96-microwell plates into 3-dimensional muscle constructs with parallel arrays of striated muscle fibers. When electrically stimulated, they generated tetanic forces measured with an automated motion tracking system. Thirty-one compounds of interest as potential treatments for patients with DMD were tested at 3 to 6 concentrations. Eleven of the compounds (insulin-like growth factor-1, creatine, beta-hydroxy-beta-methylbutyrate, trichostatin A, lisinopril, and 6 from the glucocorticoid family) significantly increased tetanic force relative to placebo-treated controls. The glucocorticoids methylprednisolone, deflazacort, and prednisone increased tetanic forces at low doses (EC(50) of 6, 19, and 56 nM, respectively), indicating a direct muscle mechanism by which they may be benefitting DMD patients. The tetanic force assay also identified beneficial compound interactions (arginine plus deflazacort and prednisone plus creatine) as well as deleterious interactions (prednisone plus creatine inhibited by pentoxifylline) of combinatorial therapies taken by some DMD patients. Since mdx muscle in vivo and DMD patients respond in a similar manner to many of these compounds, the in vitro assay will be a useful tool for the rapid identification of new potential treatments for muscle weakness in DMD and other muscle disorders.