Our inability to interpret the consequences of rare variants is an unappreciated challenge in the diagnosis of rare diseases. We developed a democratized workflow called Saturation Mutagenesis-Reinforced Functional assays (SMuRF) to inspect the direct impact variants have on enzymatic activity. We employed SMuRF to score all possible coding single nucleotide variants (SNVs) of Dystroglycanopathies-related enzyme-coding genes,FKRPandLARGE1. The utility of SMuRF scores was enhanced through the assignment of confidence scores and orthogonal assays for validation. SMuRF recapitulated and significantly expanded the knowledge gained from clinical reports and population databases, aiding in alleviating ethnic disparity in biomedical databases and improving variant classification. SMuRF expanded the training datasets of the computational predictors with the potential to improve their variant classification capability. SMuRF highlighted the critical regions in the enzyme structure which shed light on different disease mechanisms. SMuRF is the first high-throughput functional workflow to study dystroglycanopathy variants and opens the door for better variant interpretation underlying other rare diseases.