Next-generation metal-based pharmaceuticals are considered promising therapeutic agents, which may follow novel modes of action and engage with different targets compared to classical platinum(ii) anticancer agents. However, appropriate methods and assays are required to provide evidence of such unprecedented drug effects. Mass spectrometry (MS) has proved useful in probing the reactivity and selectivity of metal-based anticancer agents on a molecular level and recently also in the cellular context, especially with regard to the proteome. This chapter will discuss the design and use of competitive experiments to investigate activation pathways and binding preferences of metal-based anticancer agents by identifying reaction products via different MS setups. Moreover, cell-based approaches are described to obtain insights into novel potential targets and modes of action. Thus, mass spectrometry emerges as an enabling technology that connects molecules to mechanisms, highlighting the broad applicability of this analytical technique to the discovery and understanding of metal-based anticancer agents.