The thermal decomposition of aspartic acid (Asp, HO2CCH(NH2)CH2CO2H) on Ni surfaces occurs by a more complex pathway than on Cu surfaces where it decomposes via an aspartate species to yield CO2, H2, and CH3C≡N stoichiometrically. In addition to these three products observed on Cu, the decomposition mechanism on Ni yields CO, H2O and HC≡N. Herein, we probe the Asp decomposition mechanism on Ni(100) by X-ray photoelectron spectroscopy and by temperature programmed reaction spectra of a variety of adsorbed aspartic acid isotopomers (L-Asp, 1–13C-L-Asp, 4–13C-L-Asp, 1,4–13C2-L-Asp, 3,4–13C2-L-Asp, 1,2,3,4–13C4–15N-L-Asp, 15N-L-Asp, and D7-L-Asp). These isotope labeling experiments provide insight into the regiospecific origin within adsorbed aspartic acid of the carbon and nitrogen atoms found in the desorbing products. At low coverage, the adsorbed aspartic acid primarily undergoes inter- and intra-molecular condensation/dehydration to ketone and/or anhydride intermediates that ultimately yield CO. At higher coverages, the decomposition process is similar to that observed on Cu surfaces yielding mostly CO2 from the terminal carboxyl groups and CH3C≡N from the interior of the adsorbed aspartic acid. The continued presence of CO and H2O products at high coverages suggests the persistence of the condensation/dehydration reactions across all coverages.