Lysine l-lactylation (K_l-la) is a novel protein posttranslational modification (PTM) driven by l-lactate. This PTM has three isomers: K_l-la, N-ε-(carboxyethyl)-lysine (K_ce) and d-lactyl-lysine (K_d-la), which are often confused in the context of the Warburg effect and nuclear presence. Here we introduce two methods to differentiate these isomers: a chemical derivatization and high-performance liquid chromatography analysis for efficient separation, and isomer-specific antibodies for high-selectivity identification. We demonstrated that K_l-la is the primary lactylation isomer on histones and dynamically regulated by glycolysis, not K_d-la or K_ce, which are observed when the glyoxalase system was incomplete. The study also reveals that lactyl-coenzyme A, a precursor in l-lactylation, correlates positively with K_l-la levels. This work not only provides a methodology for distinguishing other PTM isomers, but also highlights K_l-la as the primary responder to glycolysis and the Warburg effect.