Click chemistry approaches, for example the copper-catalyzed azide-alkyne cycloaddition (CuAAC) of an azide and an alkyne, can be used for the analysis of some alkynes. This approach exploits the unique reactivity of the alkyne moiety, leading to highly specific and sensitive analyses. However, CuAAC is generally applicable only to terminal alkynes or to specially designed, highly-strained internal alkynes. Many naturally derived alkynes are internal alkynes with no ring strain, and are thus not amenable to CuAAC. According to the Human Metabolome Database, at least 173 unique internal alkyne structures are, or have the potential to be, a component of human metabolomes. These 173 internal alkynes account for 80% of the 216 alkyne-containing structures in the HMDB. More than 70% of the 173 are derived from plants or fungi. A large fraction of these come from a structurally related family of 1,3-diynes (Figure ). Natural alkynes, especially the structural clade highlighted in Figure , are often derived from fatty acids, and frequently lack moieties (amines, amides, carboxylic acids, etc.) that provide strong electrospray response during LC-MS analysis. Many are also difficult to analyze by GC-MS, because of their large size, low volatility, and/or thermal instability. Thus, poor sensitivity is another common problem during analysis of naturally occurring internal alkynes. Here we describe a specific derivatization technique for internal alkynes based on the ruthenium-catalyzed azide alkyne cycloaddition (RuAAC), a reaction previously explored for applications in organic synthesis. We have reported this concept in prior work, but here disclose expanded studies on the scope and utility of RuAAC derivatization for metabolomics.