DiscussionStatins withdrawal from the market leaded to a considerable economic burden for the pharmaceutical industry, and unravels the need to access to more relevant in vitro models dedicated to muscle damage drug discovery. In this context, CYTOO developed a physiological muscle model improving the sensitivity of myotoxic drug detection. When cultured on 2D+ Technology, primary human myoblasts faster differentiate into myotubes and present a higher degree of maturation compared to standard culture condition, i.e. a higher level of striation. Moreover, myotubes morphology is standardized which enables accessing new parameters for their characterization upon drug treatment. To further demonstrate the benefits of our model, we developed dedicated image analysis algorithms using reference toxic compounds and assess the gain of sensitivity compared to standard method thanks to a set of drugs known for their myotoxicity. Our results showed that this model is robust and compatible with High Content Screening with increased Z’ factors compared to standard assays. In addition, the normalization of myoblast differentiation into highly mature myotubes, coupled to enhanced image analysis capacities, demonstrated a higher sensitivity over standard assays and will allow the detection of myotoxic drugs during the early phases of preclinical studies for compound development.