Despite the emergence of dozens of oncogenic targets and corresponding molecularly targeted therapies, in most cases tumours continue to progress or recur due to therapeutic resistance. In the present review, we highlight the ability of MS-based phosphoproteomics to quantify oncogenic signalling networks driving tumour growth and invasion, as well as those networks enabling tumour cell survival in the presence of chemotherapeutics. Quantitative protein phosphorylation profiling will facilitate the design and development of optimal therapeutic strategies targeting the initial tumour while simultaneously blocking the predominant resistance mechanisms.