A quantitative proteomics approach was used to investigate in Oreochromis niloticus cell line across two passages P10, and P20. A cell line developed from the muscle tissue of O. niloticus (ONM) was utilized to understand the proteome in-depth. Comparative proteome analysis was performed for ONM muscle cells between two passages (10 and 20) using label free quantitative proteomics. High-resolution mass spectrometry was used for data using label-free quantification. Data were further analysed using a statistical tool across passages of rohu muscle cell line. Proteomic analysis across two passages (P10 and P20) revealed pathways involved in myogenesis were striated muscle contraction, muscle contraction, insulin pathway, regulation of actin cytoskeleton, hedgehog on state, VEGFA-VEGFR2 and signalling by WNT. The cell line generated can have wide range of application in cultivated meat as well as in in vitro application.