The effort to utilize microflow liquid chromatography for deep-scale proteomic analysis (µLC-MS/MS) is still growing. In this work, we utilized two-dimensional LC separation and peptide derivatization by tandem mass tag and assessed the capability of µLC-MS/MS to reveal protein changes associated with severe anthracycline-induced cardiomyopathy phenotype in comparison with nLC-MS/MS. Finally, control and rabbit myocardium specimens with experimentally induced anthracycline cardiomyopathy were analysed. Nearly 4000 myocardial proteins were quantified using µLC-MS/MS. In concordance with nLC-MS/MS, data evaluation revealed that severe anthracycline cardiotoxicity phenotype was associated with marked global protein dysregulation. Observed protein dysregulation consisted of profound remodelling of cardiomyocyte sarcomere as well as components of extracellular matrix. Pro-inflammatory proteins and multiple antioxidant proteins were markedly changed, whereas the levels of mitochondrial proteins were declined compared to control myocardium. In this study, µLC-MS/MS was confirmed as a versatile proteomic tool for comprehensive mapping of proteome changes related to anthracycline cardiotoxicity having a great potential to become full-fledged alternative to nLC.