<p>Electrical impedance has emerged as a powerful tool for real-time, label-free, and non-invasive monitoring of cellular processes. Here, we employed an impedance-based assay to characterize the myogenic process of control and dystrophic human myoblasts. First, we conducted a comprehensive analysis of control myoblast differentiation, assessing the effects of initial seeding density and various extracellular matrix coatings. We also evaluated the influence of electrode presence and current application, both of which improved myoblast alignment. Immortalized myoblasts from Duchenne muscular dystrophy patients exhibited marked alterations in early differentiation and maturation, which were readily detected via impedance measurements. We further compared two differentiation protocols using one control and one dystrophic representative cell line. While both protocols supported the formation of mature myotubes, impedance profiles differed depending on the culture medium. Notably, we identified the protocol with superior impedance profile reproducibility over the culture lifespan. Finally, we successfully assessed calcium homeostasis in control and dystrophic myotubes differentiated on 96-well impedance plates. Our findings underscore the potential of impedance-based assays for monitoring myogenesis and identifying disease-associated phenotypes. Moreover, 96-well impedance plates represent a robust tool for high-throughput and high-content functional analysis in muscle disease modeling and therapeutic screening.</p>