Bioorthogonal chemistry is a set of methods using the chemistry of non-native functional groups to explore and understand biology in living organisms. In this review, we summarize the most common reactions used in bioorthogonal methods, their relative advantages and disadvantages, and their frequency of occurrence in the published literature. We also briefly discuss some of the less common but potentially useful methods. We then analyze the bioorthogonal-related publications in the CAS Content Collection to determine how often different types of biomolecules such as proteins, carbohydrates, glycans, and lipids have been studied using bioorthogonal chemistry. The most prevalent biological and chemical methods for attaching bioorthogonal functional groups to these biomolecules are elaborated. We also analyze the publication volume related to different types of bioorthogonal applications in the CAS Content Collection. The use of bioorthogonal chemistry for imaging, identifying, and characterizing biomolecules and for delivering drugs to treat disease is discussed at length. Bioorthogonal chemistry for the surface attachment of proteins and in the use of modified carbohydrates is briefly noted. Finally, we summarize the state of the art in bioorthogonal chemistry and its current limitations and promise for its future productive use in chemistry and biology.