Updated project metadata. ERBB2 overexpression is associated with aggressive breast cancer (BCa) disease. The introduction in the clinic of Trastuzumab (Tz) targeting this receptor has considerably improved patient outcomes. However, de novo or acquired resistance to Tz occurs and negatively impacts prognosis. Many molecular mechanisms have been reported in the development of Tz resistance. This study aimed to establish whether common mechanisms could be identified in in vitro models of acquired BCa Tz resistance. In particular, we used widely available ERBB2+ Breast cancer cell lines BT474, MDA-MB-361, and SKBR-3, adapted in vitro to grow in Tz concentration ten times higher than the saturation one. Wt and TZ-R cells were studied to address changes in phenotype, proliferation, apoptosis, and ERBB2 membrane expression that did not highlight alterations common to the three cell lines. We used high-resolution mass spectrometry analysis to gain insight into the mechanisms associated to the adaptation of continuous growth in very high Tz concentration. This analysis identified a common set of differentially expressed proteins (DEPs) in Tz-R vs. wt cells. Bioinformatic tools available in the public domain revealed that all three Tz-R cell models shared modulation of proteins involved in the metabolism of lipids, organophosphate biosynthetic process, and macromolecule methylation. This data was partially supported by evidence of a higher number of lipid droplets in TZ-R SKBR-3 with respect to wt. In conclusion, our data strongly support previous evidence that complex metabolic adaptation, including lipid metabolism, protein phosphorylation, and possibly chromatin remodeling, may fuel Tz resistance. At the same time, identifying a common set of 10 DEPs in all three TZ-resistant cell lines may provide possible novel targets for therapeutic intervention.