Deuterium is gaining increased attention and utilization due to its unique physical and chemical properties. Deuteration has the unique benefit of positively impacting metabolic fate of pharmacologically active compounds without altering their chemical structures, physical properties, or biological activity and selectivity. In these favorable cases, deuterium substitution can in principle improve the pharmacokinetic properties and safety of therapeutic agents. The use of deuterium to create a new chemical entity not only starts with an existing drug, but can be achieved from iterative optimization in the de novo design of new compounds. Furthermore, deuterium has become a powerful tool in pharmaceutical analysis, including deuterium-labeled compounds as internal standards for extensive analysis, metabolomics, ADME, clinical pharmacology studies. This review highlights the application of deuterium in enhancing the pharmacological effects of active molecules during drug discovery and development. Additionally, deuterium-enabled pharmaceutical analysis is also covered. This review is aimed to provide references for the discovery of new deuterium-containing chemical entities with improved pharmacological properties and for the research of fate of drugs.