For amino acids used as food additives, Japan's Specifications and Standards for Food Additives stipulate the ninhydrin test as an identification test. The ninhydrin test is a simple method that involves the visual determination of purple color from the formation of Ruhemann's purple (RP) and does not require special equipment, facilitating its widespread use in society. However, because of this background, objective and molecular selective observation methods for monitoring RP itself as an analyte have not been fully investigated. Therefore, in this study, a UHPLC/MS/MS method was developed to specifically monitor RP and support visual judgment. This method identified RP-derived fragment ions at m/z 170 (ESI (-)) and 133 (ESI (+)), which can be monitored in the multiple reaction monitoring mode and were shown to correlate with the intensity of the purple color. In addition, computational chemistry was applied to scientifically estimate the molecular structures of the fragment ions. In this study, we established a useful analytical method that complements the objectivity of the ninhydrin test. This method is also expected to be utilized for further optimization of test reaction conditions.