The extracellular matrix (ECM) is dominated in-vivo by macromolecular crowding and resultant excluded volume effects [1]. It is composed of a large quantity of various macromolecules which fill the interstitial space within cells forming a hydrated gel [2]. ECM is a highly dynamic structure. It is constantly regenerated, remodeled and degraded to maintain tissue homeostasis, through the action of metalloenzymes such as collagenase, hyaluronidase (Hyal) and elastase [3]. The present investigation is part of a large multidisciplinary project, X-Crowd, aiming to scrutinize the kinetics of these enzymes in a realistic picture. For this purpose, crowded environments mimicking the ECM invitro are used. The crowding environment was simulated using dextran at two different molecular weights (40 and 476 kDa), respecting so the ratio between enzyme and crowder size. We have first studied the activity of Hyal, a glycosidase responsible for the degradation of hyaluronic acid (HA), a large polysaccharide responsible for skin hydration and cartilage lubrication. Capillary electrophoresis (CE), thanks to its miniaturized dimensions, was advantageously used to monitor the enzymatic reaction, after optimizing the injection step, taking into account the media viscosity and complexity. To better understand the effect of dextran on the catalytic activity of Hyal, a small substrate, decasaccharide (10-mers), was firstly used. Results were compared to those obtained with the high molecular weight natural substrate, HA. Moreover, the interaction between Hyal and the Dextran was characterized using microscale thermophoresis (MST), a biophysical miniaturized technique based on fluorescence detection. Hyal was thus labeled with ATTO-647 and studied in the presence of dextran with different buffer compositions and pH conditions. The inhibition study of this enzyme by a referenced inhibitor firstly, and a home-made inhibitor secondly, was also carried out in dilute and crowded media. Finally, ionic mobility spectrometry (IMS) was used to evaluate the effect of crowding on the folding state and conformational dynamics of Hyal. CE combined to IMS and MST allowed to disentangle the impact of crowding on Hyal kinetics, folding state and interactions. Therefore, this reaffirms the significance of conducting biological assays under conditions that closely mimic in vivo environments. The approach we propose ensures the development of efficient and more reliable bioactive compounds during the development process. Acknowledgement The authors acknowledge the Agence Nationale de la Recherche (ANR) for financial support of the XCrowd project (ANR-21-CE44-0020) and QUALICHIM (APR-IA-PF 2021-00149467) for financial support of ICOA. References [1] A.S. Zeiger, F.C. Loe, R. Li, M. Raghunath, K.J. Van Vliet, PLoS ONE. 2012, 7 e37904. [2] A.D. Theocharis, S.S. Skandalis, C. Gialeli, N.K. Karamanos, Adv. Drug Deliv. Rev. 2016, 97 4–27. [3] C. Chantrain, Y.A. DeClerck, Med. Sci. 2002, 18, 565–575.