底物水平磷酸化（英语：substrate-level phosphorylation）是指一类ADP或其他核苷二磷酸的磷酸化作用与底物的脱氢作用直接相偶联的反应过程。

除了发生在糖酵解和三羧酸循环之中的底物水平磷酸化之外，制造腺苷三磷酸的另一条路径是氧化磷酸化作用，这一作用发生在细胞呼吸的过程之中。在氧化磷酸化期间，还原型烟酰胺腺嘌呤二核苷酸被氧化为氧化型烟酰胺腺嘌呤二核苷酸，生成2.5分子的腺苷三磷酸，而还原黄素腺嘌呤二核苷酸被氧化时则生成1.5分子的腺苷三磷酸。氧化磷酸化利用跨线粒体膜的电化学或化学渗透质子梯度以生产腺苷三磷酸，这点是与底物水平磷酸化之间最大的不同。

Substrate-level phosphorylation is a type of metabolic reaction that results in the formation of adenosine triphosphate (ATP) or guanosine triphosphate (GTP) by the direct transfer and donation of a phosphoryl (PO₃) group to adenosine diphosphate (ADP) or guanosine diphosphate (GDP) from a phosphorylated reactive intermediate. Note that the phosphate group does not have to come directly from the substrate. By convention, the phosphoryl group that is transferred is referred to as a phosphate group.