In this project we applied a workflow for phosphoproteomics analysis to characterize the phosphorylation signalling that is triggered by the cardiotonic steroid ouabain. Ouabain is a ligand for the sodium-potassium ATPase (NKA). Low concentration of ouabain trigger oscillation of intracellular calcium concentration and modulate the phosphorylation of several signaling molecules, impacting cellular proliferation, apoptosis and cell-to-cell contacts. Monkey kidney cells (COS-7) were either treated for 10 or 20 minutes with 100 nM ouabain, or left untreated. We identify 15,348 phospho-sites, of which 1,941 are significantly regulated at either one of the time points employed. Analysis of the significantly regulated phospho-sites shed light on the molecular mechanisms by which ouabain affects cell junctions, proliferation and apoptosis.