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Caseins and ovalbumin are frequently used as wine fining agents to remove undesirable compounds like polymeric phenols. Their presence in wines is a subject of concern because may cause adverse effects on susceptible consumers, especially when their presence is not labeled. A key step for its determination is trypsin digestion, which is considered the bottleneck of bottom-up approach workflow because usually requires several hours. To reduce this time, the objective of this work was to carry out a chemometric optimization of trypsin digestion method applying infrared, microwave and ultrasound energies to determine caseins and ovalbumin in wines. The conditions of each accelerated digestion method were optimized using a Response Surface Methodology based on central composite design. The parameters optimized were digestion time and trypsin: protein ratio. The response variable evaluated was digestion yield, which was determined through the peak area of each protein transition determined by liquid chromatography-mass spectrometry. The most effective technique was microwave followed by ultrasound and infrared. Since optimal values of microwave and ultrasound-assisted digestion were the same, the later was chosen considering sample preparation and cost. Applying the proposed approach, a reduction of ca. 140 and 240-fold on digestion time was achieved compared with optimized and non-optimized conventional methods, respectively. With this workflow, both proteins were digested in a single 3 min process allowing its detection by liquid chromatography-mass spectrometry at µg L−1 level, which is ca. 60 times lower than the current limit of 0.25 mg L−1., This work is part of Jessy Pavón-Pérez thesis to obtain the degree of Doctor in Science and Analytical Technology from the University of Concepcion, Chile. Authors thank to the National Commission of Scientific and Technological Research (CONICYT) of the Chilean Government for the doctoral scholarship granted. This study was funded by the National Fund for Scientific and Technological Development (FONDECYT) Project No. 1171857 and by the National Fund for Scientific and Technological Equipment (FONDEQUIP) Project No. 130209.