Cellular dedifferentiation is required for functional regeneration in salamanders. Dedifferentiating multinucleate skeletal muscle gives rise to mononucleate cells during limb regeneration. Efficient methods and tools must be developed in order to understand the molecular cues underlying dedifferentiation. Here we describe a non-viral method to express extra-chromosomal DNA exclusively in terminally differentiated muscle without the need for cell purification steps. After cytoplasmic injection of various expression vectors into myotubes or myofibres, we detect long-lasting mRNA and protein expression in up to 70% of the injected cells. The combination of the transfection protocol with live imaging allows a time- and cost-effective screen of candidate genes in terminally differentiated muscle cells of both amphibian and mammalian origin.