Antisense oligonucleotide (ASO)-based exon skipping is a representative splice modulating therapy effective for Duchenne muscular dystrophy (DMD), a condition arising from dystrophin deficiency. For a valuable platform of evaluating exon-skipping efficacy, we previously reported a highly effective approach to convert CD90-positive human urine-derived cells (UDCs) into myotubes through the transduction of the MYOD1 gene. We anticipate that highly differentiated myotubes enable their utilization in various human muscle cell studies and a more predictive screening approach for the preclinical identification of promising ASOs at an earlier stage of the drug discovery process. Here, we describe protocols and tips for isolating UDCs, differentiating CD90-positive UDCs into myotubes, and evaluating the correction of DMD mRNA and protein levels in the myotubes after exon skipping.