Lysine L-lactylation (K_L_la) is a novel protein posttranslational modification driven by L-lactate, with three isomers: K_L_la, N-ε-(carboxyethyl)-lysine (K_ce) and D-lactyl-lysine (K_d_la). This study introduces methods to differentiate these isomers, including chemical derivatization and high-performance liquid chromatography (HPLC) analysis, and isomer-specific antibodies for high-selectivity identification. The research demonstrates that K_L_la is the primary lactylation isomer on histones and is dynamically regulated by glycolysis, while K_d_la and K_ce are observed when the glyoxalase system is incomplete. Additionally, lactyl-CoA, a precursor in L-lactylation, correlates positively with K_L_la levels. These findings highlight K_L_la as the primary responder to glycolysis and the Warburg effect, providing a methodology for distinguishing other PTM isomers.Lysine L-lactylation (K_L_la) is a novel protein posttranslational modification driven by L-lactate, with three isomers: K_L_la, N-ε-(carboxyethyl)-lysine (K_ce) and D-lactyl-lysine (K_d_la). This study introduces methods to differentiate these isomers, including chemical derivatization and high-performance liquid chromatography (HPLC) analysis, and isomer-specific antibodies for high-selectivity identification. The research demonstrates that K_L_la is the primary lactylation isomer on histones and is dynamically regulated by glycolysis, while K_d_la and K_ce are observed when the glyoxalase system is incomplete. Additionally, lactyl-CoA, a precursor in L-lactylation, correlates positively with K_L_la levels. These findings highlight K_L_la as the primary responder to glycolysis and the Warburg effect, providing a methodology for distinguishing other PTM isomers.