Epidemiological evidence indicates that diets rich in tomatoes are associated with a reduced risk of chronic diseases and certain cancers, particularly prostate cancer. The carotenoid pigment lycopene has received the most attention as a bioactive compound in reducing the risk of prostate cancer, but a causal relationship has not yet been established. Consumption of processed tomato products has been associated with an even lower risk of prostate cancer than fresh tomatoes, indicating the potential importance of processing-induced compounds in cancer prevention. The Maillard reaction occurs during food processing in tomatoes and other foods and may be a source of bioactive compounds. Amadori compounds are formed as an intermediate in this reaction and their production is affected by temperature, time, pH, and water activity during processing. One particular Amadori compound, fructose-histidine, or FruHis, was recently shown to work in concert with lycopene in processed tomatoes in vitro and in vivo to further reduce the risk of prostate cancer in animal studies. However, very little is known about the formation of FruHis and other Amadori compounds during food processing. In addition, comprehensive methods for the separation, identification, and quantification of FruHis and Amadori compounds are lacking. In this study we set out to develop a hydrophilic interaction liquid chromatography (HILIC)-ESI-MS/MS method for the analysis of FruHis and Amadori compounds and to use this method to analyze the formation of FruHis in processed tomato products. A HILIC method using formic acid and acetonitrile was developed on an amide column to successfully separate these compounds and was particularly suited for quantification of FruHis. This method was used to quantify FruHis during typical tomato paste processing. The FruHis content increased during tomato paste concentration, reaching a maximum of 3.5 mg per 100 g dry paste before decreasing during retorting due to degradation to colored Maillard products. The initial formation of FruHis was tied to a key combination of heat applied and water lost as measured by percent soluble solids. The content of FruHis in a number of commercial juices, sauces, pastes, and powders ranged from 1.05 to 12.31 mg per 100 g, with tomato powders followed by tomato pastes containing the highest concentrations. The results of this work will provide a basis for future studies on the biological activity of FruHis and Amadori compounds and may one day lead to the development of a FruHis-rich tomato-based functional food for cancer prevention.