In biochemistry, phosphorylation is described as the "transfer of a phosphate group" from a donor to an acceptor or the addition of a phosphate group to a molecule. A common phosphorylating agent (phosphate donor) is ATP and a common family of acceptor are alcohols:

This equation can be written in several ways that are nearly equivalent that describe the behaviors of various protonated states of ATP, ADP, and the phosphorylated product. As is clear from the equation, a phosphate group per se is not transferred, but a phosphoryl group (PO₃^-). Phosphoryl is an electrophile. This process and its inverse, dephosphorylation, are common in biology. Protein phosphorylation often activates (or deactivates) many enzymes.

Phosphorylation is essential to the processes of both anaerobic and aerobic respiration, which involve the production of adenosine triphosphate (ATP), the "high-energy" exchange medium in the cell. During aerobic respiration, ATP is synthesized in the mitochondrion by addition of a third phosphate group to adenosine diphosphate (ADP) in a process referred to as oxidative phosphorylation. ATP is also synthesized by substrate-level phosphorylation during glycolysis. ATP is synthesized at the expense of solar energy by photophosphorylation in the chloroplasts of plant cells.

Phosphorylation of sugars is often the first stage in their catabolism. Phosphorylation allows cells to accumulate sugars because the phosphate group prevents the molecules from diffusing back across their transporter. Phosphorylation of glucose is a key reaction in sugar metabolism. The chemical equation for the conversion of D-glucose to D-glucose-6-phosphate in the first step of glycolysis is given by:

Glycolysis is an essential process of glucose degrading into two molecules of pyruvate, through various steps, with the help of different enzymes. It occurs in ten steps and proves that phosphorylation is a much required and necessary step to attain the end products. Phosphorylation initiates the reaction in step 1 of the preparatory step (first half of glycolysis), and initiates step 6 of payoff phase (second phase of glycolysis).

Glucose, by nature, is a small molecule with the ability to diffuse in and out of the cell. By phosphorylating glucose (adding a phosphoryl group in order to create a negatively charged phosphate group), glucose is converted to glucose-6-phosphate, which is trapped within the cell as the cell membrane is negatively charged. This reaction occurs due to the enzyme hexokinase, an enzyme that helps phosphorylate many six-membered ring structures. Phosphorylation takes place in step 3, where fructose-6-phosphate is converted to fructose 1,6-bisphosphate. This reaction is catalyzed by phosphofructokinase.

While phosphorylation is performed by ATPs during preparatory steps, phosphorylation during payoff phase is maintained by inorganic phosphate. Each molecule of glyceraldehyde 3-phosphate is phosphorylated to form 1,3-bisphosphoglycerate. This reaction is catalyzed by glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The cascade effect of phosphorylation eventually causes instability and allows enzymes to open the carbon bonds in glucose.

Phosphorylation functions is an extremely vital component of glycolysis, as it helps in transport, control, and efficiency.