LC-MS is a powerful technique in biomolecular research. In addition to its uses as a tool for protein and peptide quantization, LC-MS can also be used to quantify the activity of signalling and metabolic pathways in a multiplex and comprehensive manner, i.e. as an 'activitomic' tool. Taking cancer research as an illustrative example of application, this review discusses the concepts of biochemical pathway analysis using LC-MS-based proteomic and activitomic techniques.