Hydrolysis (/haɪˈdrɒlɪsɪs/; from Ancient Greek hydro- 'water' and lysis 'to unbind') is any chemical reaction in which a molecule of water breaks one or more chemical bonds. The term is used broadly for substitution and elimination reactions in which water is the nucleophile.

Biological hydrolysis is the cleavage of biomolecules where a water molecule is consumed to effect the separation of a larger molecule into component parts. When a carbohydrate is broken into its component sugar molecules by hydrolysis (e.g., sucrose being broken down into glucose and fructose), this is recognized as saccharification.

Hydrolysis reactions can be the reverse of a condensation reaction in which two molecules join into a larger one and eject a water molecule. Thus hydrolysis adds water to break down molecules, whereas condensation joins molecules through the removal of water.

Hydrolysis is a chemical process in which a molecule of water is added to a substance, causing both the substance and water molecule to split into two parts. In such reactions, a chemical bond is broken, with one fragment of the target molecule (or parent molecule) gaining a hydrogen ion, and the other gaining a hydroxide. In living systems, most biochemical reactions (including ATP hydrolysis) take place during the catalysis of enzymes. The catalytic action of enzymes allows for the hydrolysis of proteins, fats, oils, and carbohydrates.

Ester and amide hydrolysis occurs through nucleophilic acyl substitution where water acts as a nucleophile (a nucleus-seeking agent, e.g., water or hydroxyl ion), attacking the carbon of the carbonyl group of the ester or amide. Under acidic conditions, the carbonyl group is activated via protonation, allowing for direct nucleophilic attack by water. In an aqueous base, hydroxyl ions are better nucleophiles than polar molecules such as water due to the negative charge localized on the oxygen and therefore directly attack the carbonyl group.

Upon hydrolysis, an ester is converted into a carboxylic acid plus an alcohol, while an amide converts into a carboxylic acid and an amine or ammonia (which in the presence of acid are immediately converted to ammonium salts). One of the two oxygen groups on the carboxylic acid are derived from a water molecule and the amine/ammonia or alcohol gains the hydrogen ion.

Perhaps the oldest commercially practiced example of ester hydrolysis is saponification (formation of soap). It is the hydrolysis of a triglyceride (fat) with an aqueous base such as sodium hydroxide (NaOH). During the process, glycerol is formed, and the fatty acids react with the base, converting them to salts. These salts are called soaps, commonly used in households. Under biological conditions, this reaction is catalyzed by lipases for the digestion of fats, acting when adsorbed to an oil-water interface. Other esterases function in water, serving a variety of biological functions.

A key biological application of amide hydrolysis is the digestion of proteins into amino acids. Proteases, enzymes that aid digestion by causing hydrolysis of peptide bonds in proteins, catalyze the hydrolysis of peptide bonds in peptide chains, releasing polypeptide fragments two to six amino acids long. Those fragments are then broken down into single amino acids via carboxypeptidases secreted by the pancreas.