A quantitative proteomics approach was used to investigate in Labeo rohita cell line across three passages P1, P15 and P25. A new cell line developed from the muscle tissue of L. rohita (LR) was utilized to understand the proteome in-depth. Comparative proteome analysis was performed for LR muscle cells at different passages (1, 15 and 25) 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. Functional analysis of differentially expressed proteins was marked as glycolytic (eno1 and eno3), metabolic (pgm, smyd1, ak1, aldob and bpgm), cytoplasmic ribosomal (rpl27, rpl3 and eifa5) and carbon metabolic (pdh) pathways across the three passages. The proteins observed across the pathways were also correlated with the differentiation of LR muscle cells during myogenesis. The current study provides insight to determine the different groups of proteins that are expressed during the development of myotubes.