The evaluation of binding affinities between large biomolecules and small ligands is challenging and requires highly sensitive techniques. Microscale thermophoresis (MST) is an emerging biophysical technique used to overcome this limitation. This work describes the first MST binding method to evaluate binding affinities of small ligands to lipases from crude porcine pancreatic extracts. The conditions of the MST assay were thoroughly optimized to successfully evaluate the dissociation constant (Kd) between pancreatic lipases (PL) and triterpenoid compounds purified from oakwood. More precisely, the fluorescent labeling of PL (PL*) using RED-NHS dye was achieved via a buffer exchange procedure. The MST buffer was composed of 20 mM NaH2PO4 + 77 mM NaCl (pH 6.6) with 0.05% Triton-X added to efficiently prevent protein aggregation and adsorption, even when using only standard, uncoated MST capillaries. Storage at -20°C ensured stability of PL* and its fluorescent signal. MST results showed that crude pancreatic extracts were suitable as a source of PL for the evaluation of binding affinities of small ligands. Quercotriterpenoside-I (QTT-I) demonstrated high PL* binding affinity (31 nM) followed by 3-O-galloylbarrinic acid (3-GBA) (500 nM) and bartogenic acid (BA) (1327 nM). To enrich the 50 kDa lipase responsible for the majority of hydrolysis activity in the crude pancreatic extracts, ammonium sulfate precipitation was attempted and its efficiency confirmed using capillary electrophoresis (CE)-based activity assays and HRMS. Moreover, to accurately explain enzyme modulation mechanism, it is imperative to complement binding assays with catalytic activity ones.< Réduire