Cancer remains a significant global health challenge, necessitating the development of innovative and cost-effective treatments. Drug repurposing, particularly of cardiac glycosides (CGs), offers a promising strategy to expedite the discovery of anti-tumor drugs. This study employs proteome integral solubility alteration (PISA) to investigate the multifaceted mechanisms of action of CGs, including digoxin, digitoxin, and ouabain. Our study revealed that CGs may interact with a spectrum of proteins associated with cellular metabolism, ribosomal function, and apoptosis, suggesting a complex interplay between these pathways in cancer cells. Besides, it suggests that CGs could be repurposed to target cancer through a multifaceted approach that disrupts cellular metabolism, protein synthesis, and mitochondrial function. Further studies are warranted to confirm the functional implications of these interactions and to explore the therapeutic potential of CGs in cancer treatment.