Myogenesis is a highly regulated multi-step process involving myoblast proliferation and differentiation. Although studies over the last decades have identified several factors governing these distinct major phases, many of them are not yet known. In order to identify novel genes, we took advantage of the C2C12 myoblastic line to establish a functional siRNA screen combined with quantitative-imaging analysis of a large amount of differentiated myoblasts. We knocked down 100 preselected mouse genes without a previously characterized role in muscle. Using image analysis, we tracked gene-silencing phenotypes by quantitative assessment of cellular density, myotube quantity, myotube morphology and fusion index. Our results have revealed six genes involved in both stages of C2C12 myogenesis and 13 genes specific to the differentiation stage. These findings prove that our RNAi-based screen could be an efficient tool to detect clear and subtle phenotypes allowing the identification of new myogenic regulators in mammals.