A fast and simple high-performance liquid chromatography method suitable for determining furosine level in heat-treated food samples was developed. The analysis of furosine was performed by a novel mixed-mode column that provides multiple and simultaneous retention mechanisms including cation-exchange, anion-exchange, reversed-phase, or hydrophilic interaction. Each retention mechanism could be independently controlled by setting chromatographic conditions. Adequate retention and selectivity of polar charged furosine were achieved by adjusting mobile phase pH, buffer concentration, organic content, and ionic strength. The optimized method was successfully applied to determinate furosine in durum wheat semolina pasta samples. Furosine level in pasta may be used as a reliable marker of health and nutritional damage occurring during pasta manufacture. Indeed, a low content of furosine is generally related to high nutritional quality of food and application of mild heat treatments. A wide range of dry pasta samples, collected from both supermarkets (large-scale retail trade) and shops selling local products, were analyzed. Variable amounts of furosine, ranging from 107 to 506 mg/100 g of protein, were found in pasta samples. The proposed method allows to discriminate products submitted to different time–temperature conditions during the drying process. At the same time, it may be used to highlight potential label fraud.