Nuclear positioning plays important roles for certain cellular functions. This is particularly relevant in skeletal muscle cells also known as myofibers in which nuclear positioning defects were shown to hinder muscle function. Myofibers are multinucleated cells with nuclei equally distributed at the periphery of the cell. However, nuclei can be found centrally located during myogenesis before anchoring at the periphery or in certain muscle disorders, either due to regenerating myofibers or defects in nuclear movement. As such, nuclear localization in myofibers (central or peripheral) can be used to assess myofiber maturity, regeneration, or health. To study how nuclei reach the periphery of myofibers during development, we devised a unique protocol to mature myofibers thereby recapitulating later stages of differentiation, including nuclear movement to the periphery. Here we describe how to use this system to study nuclear positioning and other nuclear characteristics such as nuclear stiffness or rupture.