We utilised Super-SILAC mouse technology to perform an unbiased, quantitative analysis of the cardiac phosphoproteome in mice acutely treated in vivo with either saline or isoprenaline (ISO), a non-selective β-AR agonist. Using a customized mass spectrometry-based workflow (Figure 1), our study identified hundreds of phosphosites that are significantly regulated by β-AR stimulation, including both novel phosphoproteins and phosphosites that have not been previously identified as downstream effectors of β-AR stimulation and established downstream components of cAMP/PKA and CaMKII signaling pathways. Interestingly, our study revealed both up- and down-phosphorylation in comparable numbers, indicative of an intricate signaling network activated in response to β-AR stimulation.