Mass spectrometry (MS)-based proteomics has been used to study global and dynamic cell signalling for over a decade and phosphoproteomics has become a burgeoning field in its own right. Developments over the last few years have steadily increased the scope of phosphoproteomics studies in molecular biology, but major challenges remain. Chief among these are practical limitations in scaling, performance and reproducibility. However, these limitations are now falling away, with the development of robust and reproducible workflows aimed at simplifying phosphoproteomics experiments. The ‘EasyPhos’ method now enables the rapid and accurate acquisition of large numbers of phosphoproteomes, facilitating the analysis of dynamic and in vivo signalling. Very recent developments to the workflow now ensure high performance in sample-limited conditions, while simultaneously reducing sample preparation time. For example, protein precipitation steps have been eliminated, reducing opportunities for sample loss and variability. Together with sensitivity-boosting optimisations this has yielded a 300% improvement in performance of the method under sample limited conditions. Collectively this now enables experiments from 200 μg of cell lysate for a depth of >10,000 quantified phosphopeptides in 1 h of MS measurement time. I have recently applied these technologies to wide ranging biological questions, including the investigation of GPCR Kappa Opioid signalling in both a time- and brain-region resolved manner, and unravelling the temporal complexity of stem cell differentiation. The Kappa Opioid study has provided the first systems-view of kappa opioid receptor (KOR) signalling in vivo, revealing new mechanisms of drug action. In mice treated with KOR agonists, we identified a novel mechanism to abolish the major known side effect of aversion while preserving beneficial therapeutic analgesic and anticonvulsant effects. Excitingly, these and several other studies are collectively revealing unprecedented regulation of the phosphoproteome, emphasising the central role of post-translational regulation in proteome plasticity.