The great research interest in the quantification of reactive carbonyl compounds (RCCs), such as methylglyoxal (MGO) in biological and environmental samples, is reflected by the fact that several publications have described specific strategies to perform this task. Thus, many reagents have also been reported for the derivatization of RCCs to effectively detect and quantify the resulting compounds using sensitive techniques such as liquid chromatography coupled with mass spectrometry (LC-MS). However, the choice of the derivatization protocol is not always clear, and a comparative evaluation is not feasible because detection limits from separate reports and determined with different instruments are hardly comparable. Consequently, for a systematic comparison, we tested 21 agents in one experimental setup for derivatization of RCCs prior to LC-MS analysis. This consisted of seven commonly employed reagents and 14 similar reagents, three of which were designed and synthesized by us. All reagents were probed for analytical responsiveness of the derivatives and stability of the reaction mixtures. The results showed that derivatives of 4-methoxyphenylenediamine and 3-methoxyphenylhydrazinereported here for the first time for derivatization of RCCsprovided a particularly high responsiveness with ESI-MS detection. We applied the protocol to investigate MGO contamination of laboratory water and show successful quantification in a lipoxidation experiment. In summary, our results provide valuable information for scientists in establishing accurate analysis of RCCs.