The study was performed to analyze the proteomic profiling of doxorubicin-treated H9c2 cardiomyocytes in order to identify novel protein biomarkers associated with doxorubicin-induced cardiomyopathy. The protein profiling of H9c2 cells in response to doxorubicin at an apoptosis-induced concentration of 0.5 μM were compared using iTRAQ analysis. Western-blot analysis was used to confirm differentially expressed proteins identified in the proteomic study. A total of 22 differently expressed proteins were identified in doxorubicin-treated H9c2 cells including 15 up-regulated and 7 down-regulated proteins. Gene Ontology (GO) analysis revealed that 10 altered proteins were enriched in the process of apoptosis. We further validated the expression of cathepsin B and its possible regulator nuclear factor kappa B (NF-κB) in H9c2 cells were increased during doxorubicin treatment using Western-blots. Differentially expressed proteins might provide clues to clarify novel mechanisms underlying doxorubicin-induced cardiomyopathy. Our results also suggest that increased cathepsin B expression might be associated with NF-κB up-regulation, and the exact mechanisms need to be clarified.