In this doctoral thesis, the focus lied on identifying the protein targets of different bioactive cysteine-reactive small molecules using chemical proteomics. It is subdivided in three main chapters, each chapter covers the process of target identification of a single molecule. In the first part, SPTSSB as a target of the natural product gambogic acid and an analog of it was identified. Treatment of cells and mice using these compounds leads to a decrease in the important signaling molecule sphingosine-1-phosphate. In the second part, POLE3 was identified as a target of a sesquiterpenoid derived from the fir tree genus Abies. Engagement of this protein led to chemosensitization to the DNA damaging drug etoposide and might be used in future as a new strategy for treating cancer. In the final part, screening of a small library of ethynyl benziodoxolone reagents led to the discovery of a compound that binds to the interesting protein targets GAPDH and GLO1, both involved in cancer progression.