During glycolysis, the net gain of ATP is 2 ATP molecules per glucose molecule. Glycolysis is a series of biochemical reactions that break down one glucose molecule 6 carbons into two molecules of pyruvate 3 carbons each . The process occurs in the cytoplasm of cells and does not require oxygen.The generation of ATP in glycolysis occurs in two main steps:1. Energy investment phase: In this phase, 2 ATP molecules are consumed to phosphorylate glucose and its intermediate, fructose-6-phosphate. This results in the formation of fructose-1,6-bisphosphate, which is then split into two 3-carbon molecules, glyceraldehyde-3-phosphate G3P and dihydroxyacetone phosphate DHAP . DHAP is converted into G3P, so there are two G3P molecules entering the next phase.2. Energy payoff phase: In this phase, each G3P molecule undergoes a series of reactions that generate ATP and NADH. First, G3P is oxidized, and inorganic phosphate Pi is added to form 1,3-bisphosphoglycerate. During this reaction, NAD+ is reduced to NADH. Then, a high-energy phosphate group is transferred from 1,3-bisphosphoglycerate to ADP, forming ATP and 3-phosphoglycerate. This process is called substrate-level phosphorylation. Finally, the remaining reactions convert 3-phosphoglycerate to pyruvate.Since there are two G3P molecules entering the energy payoff phase, a total of 4 ATP molecules are generated 2 ATP per G3P . However, considering the 2 ATP molecules consumed in the energy investment phase, the net gain of ATP during glycolysis is 2 ATP molecules per glucose molecule.