The function and activity of proteins are directly related to their higher order structure (HOS) since the three-dimensional arrangement of the sequence determines which residues are close in space. A well-studied example of this relationship is the digestive enzyme α-chymotrypsin, that differs only by four amino acids from its inactive precursor. Yet, the cleavage of these four residues is known to initiate small but important conformational changes which give rise to activation of the enzyme. Characterizing those changes in proteins through 3D-structural analysis requires tremendous effort and crystal structures are often not representative for the protein structure in the solution of interest. Alternatively, the secondary structure can be used as a sensitive reporter for changes in the overall protein arrangement. Here, we employ Microfluidic Modulation Spectroscopy (MMS), an automated quantum cascade laser (QCL)-based infrared technique, to determine the structural changes along the activation of α-chymotrypsin in solution. Using the example of chymotrypsin, the goal of this study is to show that conformational changes in proteins which may substantially impact their function, can be successfully monitored with MMS.