Casein and ovalbumin are proteins commonly used as wine fining agents that may trigger allergic reactions in susceptible individuals. Therefore, their occurrence in wines could become a health risk, moreover when their presence is not reported. The objective of this work was to determine the occurrence of casein and ovalbumin in Chilean wines. Proteins were extracted by ultrafiltration and digested applying an optimised ultrasound-assisted method. Peptides were quantified by validated tandem mass spectrometry method using stable isotope dilution analysis. Optimal digestion was achieved in 3 minutes at a 1:10 enzyme protein ratio. The method was validated following ICH guidelines showing determination coefficients R ≥ 0.99, repeatability, and intermediate precision with RSD values <1.95% and recoveries from 89.8% to 115.1% (RSD < 5.84%). The method was applied to analyse 60 wine samples. Fifty-six samples showed quantifiable levels, from which 28% presented a total casein and ovalbumin concentration equal or higher to the European limit (0.25 mg L).