Lysophosphatidic acid (LPA) is an endogenous cell signaling molecule, and dysregulation of LPA signaling pathways is accompanied by several types of cancer. Herein, we developed a chemical proteomic method for the proteome-wide identification of LPA-binding proteins. The method involves the synthesis of a desthiobiotin-conjugated LPA acyl phosphate probe for the covalent labeling, enrichment, and subsequent LC-MS/MS identification of LPA-binding proteins at the proteome-wide level. By conducting labeling reactions at two different probe concentrations (10 and 100 μM) in conjunction with an SILAC (stable isotope labeling by amino acids in cell culture)-based workflow, we characterized the LPA-binding capabilities of these proteins at the entire proteome scale, which led to the identification of 86 candidate LPA-binding proteins in HEK293T cells. Moreover, we validated that two of these proteins, annexin A5 and phosphoglycerate kinase 1, can bind directly with LPA. Together, we developed a novel LPA probe for the identification and characterizations of LPA-binding proteins from the entire human proteome. The method should be adaptable for the identification of other lipid-binding proteins.