Biological selection makes natural products promising scaffolds for drug development and the ever growing number of newly identified, structurally diverse molecules helps to fill the gaps in chemical space. Elucidating the function of a small molecule, such as identifying its protein binding partners, its on- and off-targets, is becoming increasingly important. Activity- and affinity-based protein profiling are modern strategies to acquire such molecular-level information. Introduction of a molecular handle (azide, alkyne, biotin) can shed light on the mode of action of small molecules. This Concept article covers central points on synthetic methodology for integrating a terminal alkyne into a molecule of interest.