Limb-girdle muscular dystrophy type 2A (LGMD2A) is an autosomal recessive muscular disorder caused by mutations in the gene coding for calpain 3, a calcium-dependent protease. In order to improve LGMD2A diagnosis, we have developed an in vitro assay that can detect the proteolytic activity of calpain 3 in a muscle sample. This assay is based on the use of an inactive calpain 3 as a substrate for active calpain 3 molecules. A total of 79 human biopsies consisting of 42 confirmed LGMD2A and 37 other samples (normals, others myopathies and undiagnosed muscles) have been analyzed. Proteolytic activity was either reduced or absent in 68% of LGMD2A biopsies. In the remaining 32%, normal proteolytic activity was found despite the presence of calpain 3 mutation(s), suggesting that other calpain 3 properties, possibly in vivo recognition of physiological substrates or correct calpain 3 localisation in muscle fiber, might be impaired to give rise to the LGMD2A phenotype.