Limb Girdle Muscular Dystrophy type 2i (LGMD2i) is an autosomal recessive disease caused by mutations in the fukutin-related protein (FKRP) gene. The Majority of FKRP mutations lead to LGMD2i which is the third most common form of Limb Girdle Muscular Dystrophy (LGMD). All FKRP related diseases result in the absence or reduction of O-glycosylation of alpha dystroglycan (a-DG). This results in the disruption of sarcolemma integrity and leads to contraction induced damage, inflammation and loss of muscle mass and function. Currently there are no directed therapies for the treatment of LGMD2i. We have established a multiplexed western blot-based method that reports on the relative O-glycosylation of alpha dystroglycan from muscle biopsies. Here we report the results on 13 LGMD2i patients representing homozygous and complex heterozygous mutations in FKRP. In all patients there is decreased matriglycan expression as compared to healthy human donor tissue. Four of these patients have longitudinal biopsies and there is little change in matriglycan expression from the initial baseline biopsy measurement but still significantly reduced as compared to donor tissue. The semi-quantitative nature of this method may be of use in understanding the longitudinal changes in patients with LGMD2i and in assessing the impact of therapeutics directed at increasing the O-glycosylation of alpha dystroglycan.