In chemistry, the term substrate is highly context-dependent. Broadly speaking, it can refer either to a chemical species being observed in a chemical reaction, or to a surface on which other chemical reactions or microscopy are performed.

In the former sense, a reagent is added to the substrate to generate a product through a chemical reaction. The term is used in a similar sense in synthetic and organic chemistry, where the substrate is the chemical of interest that is being modified. In biochemistry, an enzyme substrate is the material upon which an enzyme acts. When referring to Le Chatelier's principle, the substrate is the reagent whose concentration is changed.

In the latter sense, it may refer to a surface on which other chemical reactions are performed or play a supporting role in a variety of spectroscopic and microscopic techniques, as discussed in the first few subsections below.