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. A total of 138 proteins containing ≥ two unique peptides were used to assess expression at the different passages. Hierarchical clustering of the top 25 proteins with significant differences in abundances was plotted across the different passages with a fold-change of 1.5. Protein-protein interactions of significantly expressed proteins with respective KEGG pathways were represented using STRING. The metascape depicts the network of significantly enriched proteins with functional annotation of protein-encoding genes. 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 expression of ribosomal proteins like eif5a, rps8a, rpl30, rpl27, rpl3 in the LR muscle cells participates in muscle growth and maintenance by altering translational capacity of the cells. Cytoskeletal proteins such as annexin and tropomyosin are involved in the adhesion and reorganization of cytoskeleton signaling during early differentiation of LR muscle cells. 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. Newly developed muscle cell line provides a benchmark for understanding in vitro myogenesis towards cultivated meat production.