The extracellular matrix (ECM) is a complex network of macromolecules that play essential structural and functional roles in tissue organization and the regulation of cellular processes. It undergoes continuous remodeling, primarily mediated by matrix metalloproteinases (MMPs), serine proteases (e.g., human neutrophil elastase, HNE), and hyaluronidases (Hyal). However, these enzymes also contribute to pathological tissue remodeling, leading to conditions such as inflammation, tumor progression, and metastasis. Understanding their molecular characteristics and detailed mechanisms, and developing potential inhibitors is a significant challenge. Despite growing interest, there are only a few studies detailing their protein structures, spatial conformations, oligomerization, physicochemical properties (e.g., the exact pI value for Hyal), or the relationship between their catalytic activity and preparation/analysis conditions. Additionally, the literature lacks information regarding the quality or purity of samples used in catalytic and affinity assays, and the impact of sample quality on in vitro results. This internship project utilizes highly complementary analytical tools. Quality control by UHPLC-ESI-QTOF MS with online desalting and MALDI-TOF MS confirmed the good quality of HNE, MMP-1, and Hyal2 preparations but revealed that BTH is surprisingly impure. Proteomics assays even detected the presence of BSA in BTH samples. Native Liquid MALDI successfully characterized the oligomerization of HNE, MMP-1, and Hyal2. Results indicated that HNE does not form oligomers, MMP-1 forms dimers, and Hyal2 forms tetramers. Furthermore, a significant influence of a crowding agent (dextran 476 kDa) used to mimic the ECM on the inhibition of BTH by both natural and synthetic compounds was demonstrated by CE-UV catalytic assays and by MST affinity tests. This finding underscores the importance of studying proteins under conditions that closely resemble physiological environments. The innovative information collected during this internship will significantly advance studies on ECM targets.