As one of the most important platinum drugs, the interactions of cisplatin with proteins play very important roles in its anticancer action and side effects. Here, the interaction of cisplatin with cytochrome c was investigated using Fourier transform ion cyclotron resonance mass spectrometry (FTICR MS). On the basis of the high-resolution data, cytochrome c-Pt(NH(3))Cl was found to be the primary monoadduct. The platinated monoadducts were related to molar ratios of cytochrome c and cisplatin, and corresponding reaction pathways were proposed. Multiple binding sites of cisplatin on cytochrome c were directly determined by FTICR MS combined with trypsin digestion without liquid chromatography (LC) separation. Four binding sites (Met65, Met80, His18, and His33) for cisplatin on cytochrome c were identified. Moreover, hydrogen/deuterium exchange (H/DX) combined with FTICR MS provides the sensitive method to insight the small conformation change of cytochrome c induced by cisplatin. This strategy can be applied to comprehensive investigation of metallodrug/protein complexes.