The greater awareness of consumers regarding the sustainability
of food chains has shifted part of the consumption from animal protein
sources to vegetable sources. Among these, of relevance both for human
food use and for animal feed, is soy. However, its high protein content
is unfortunately accompanied by the presence of antinutritional factors,
including Kunitz’s trypsin inhibitor (KTI). Now there are few
analytical methods available for its direct quantification, as the
inhibitory activity against trypsin is generically measured, which
however can be given by many other molecules and undergo numerous
interferences. Therefore, in this work, a direct label-free liquid
chromatography–mass spectrometry (LC–MS) method for
the identification and quantification of trypsin Kunitz inhibitor
KTI3 in soybean and derivative products has been developed. The method
is based on the identification and quantification of a marker peptide,
specific for the protein of interest. Quantification is achieved with
an external calibration curve in the matrix, and the limit of detection
and the limit of quantification of the method are 0.75 and 2.51 μg/g,
respectively. The results of the LC–MS method were also compared
with trypsin inhibition measured spectrophotometrically, highlighting
the complementarity of these two different pieces of information.