Enzymatic post-translational modifications (PTMs) regulate the structure, function, and stability of proteins that are involved in a myriad of vital cellular processes. Incorporation of these modifications onto substrate proteins creates additional complexity to the already expansive proteome, which further hinders efforts for complete functional annotation. Importantly, dysregulation to the activity of PTMs has been implicated in various pathologies such as inflammatory, autoimmune, cardiovascular, and neurodegenerative diseases as well as several types of cancers. Over recent decades, advancements to the experimental techniques employed to study post-translational modifications have considerably expanded the understanding of the functional role they impart on biological processes on both a single-protein basis and a global level. This chapter first provides an overview of the prominent PTM types with a focus on their respective enzymatic regulators, substrate proteins, and their roles in the onset and progression of cancer. The current experimental approaches used to study PTMs such as chemical labeling, unnatural amino acid incorporation, biophysical-based imaging, and mass spectrometry-based proteomics are also highlighted along with a brief discussion of their limitations. Finally, the current trends within the field and a perspective on future work are presented.