Amino acid derivative reactivity assay (ADRA) is an in chemico assay for assessing the skin sensitization potential of chemicals by evaluating the reactivity of nucleophilic reagents that mimic skin proteins. N-(2-(1-Naphthyl)acetyl)-l-cysteine (NAC) and α-N-(2-(1-naphthyl)acetyl)-l-lysine (NAL), used as nucleophilic reagents, are small-molecule derivatives of two different amino acids, each with a naphthalene ring attached. The rate of decrease in the amount of NAC or NAL in the reaction solution is evaluated in this assay as an indicator of the test substance's skin sensitization ability. However, the products formed between the nucleophilic reagent and the test substance, which play an important role in vivo, are not directly identified. Therefore, six highly reactive chemicals, including the proficiency substances listed in the OECD Test Guidelines─squaric acid diethyl ester, 2-methyl-2H-isothiazol-3-one (MI), p-benzoquinone, palmitoyl chloride, diphenylcyclopropenone (DPCP), and imidazolidinyl urea (IU)─were used to determine each formed product. Samples were prepared according to the standard ADRA method, and the formed products were predicted on the basis of the reaction mechanism. Excluding DPCP, the estimated structures were validated using mass spectrometry and nuclear magnetic resonance spectrometry on the synthesized samples. In this manner, the products of each nucleophile were confirmed for all examined test substances. The estimated structure products were obtained through a series of reactions initiated by the nucleophilic attack of NAC's thiol group or NAL's amino group on the test substance's electron-deficient carbonyl carbon. However, contrary to expectations, disulfide-linked-type ring-opened products were detected in the case of MI, and products with free formaldehyde in solution were detected in the case of IU. In summary, all skin sensitizers tested herein reacted with NAC and/or NAL to give products. This supports the theoretical validity of ADRA, which provides an indirect evaluation of the formed products based on a decrease in nucleophilic reagents.