The chemical composition of polymers (i.e., types of end groups, co-polymer ratio, molecular weight) is an important parameter for determining their mechanical properties, including degradability and melting point. A detailed understanding of polymer composition is pivotal to designing materials that offer improved recyclability and controlled biodegradation. Therefore, the development of catalytic methods that enable controlled polymer depolymerization is of high interest for applications in polymer recycling and analytical chemistry that aim to characterize polymer distribution. The physical and chemical degradation of polymers includes thermal, photochemical, mechanochemical, and oxidating processes. These processes can successfully degrade the polymer but lack selectivity. In contrast, enzymes offer the potential for chemoselective degradation of polymers by targeting specific bonds. Moreover, enzymes can display different hydrolytic kinetics for different monomers depending on the neighboring monomer within the polymer sequence. Therefore, available and novel hydrolases could be applied to characterize the microstructure of synthetic polymers similarly to what is done for the characterization of proteins (i.e., chemoselective digestion). The capability of some hydrolases to degrade synthetic polyesters has been investigated over the past two decades, leading to the discovery and engineering of variants that can fully degrade polyethylene terephthalate (PET) polyester polymer and, therefore, potentially be used for recycling and bioremediation purposes. The enzymes used for this purposes were cutinases isolated from microbial strains (e.g. Ideonella sakaiensis and Thermobifida fusca) or fungi (Fusarium solani, Humicola insolens, and Aspergillus oryzae), and further engineered. Among the recently published candidates enzymes for PET hydrolysis are Thermobifida fusca cutinase (TfCut2) , Thermobifida cellulosilytica cutinase 1 (ThcCut1) and an engineered leafbranch compost cutinase (LCC WCCG ). Further recent research has also led to the discovery and engineering of other catalytically active and thermostable enzymes for the depolymerization of PET. However, these recent works on polyester degradation by enzymes has mainly focused on methods to degrade homopolymers back to the starting monomers. Müller et al. have studied the process of co-polyesters hydrolysis, revealing that the cleavage rates are dependent on the heterogeneity of the polymer as well as the chemical structure and molecular environment (crystallinity) in which the ester bonds are embedded. Yet, to date, analytical investigations studying the changes of chemical structures in co-polymers as a result of enzymatic degradation are lacking. Herein, we report the development and use of cutinases to depolymerize model polyester homopolymers and a copolymer. Two enzymes were investigated for their selectivity in the hydrolysis of small molecules and the depolymerization of structurally diverse polyester (i.e., homopolymers and copolymers). One of these enzymes was engineered from the cutinase family to improve its thermostability and allow depolymerization reactions at higher temperatures in aqueous buffer media. The other studied cutinase was a previously reported variant from the leaf-branch compost cutinase (LCC). Various LC and LC-MS methods were applied to characterize the products and the remaining polymer after the enzymatic hydrolysis. An overview of the steps performed is presented in SI, Fig. . As polyesters, we selected polymers with different physical and chemical properties composed by different monomers (Fig. ): polyester 1 (PES1) (green box) as an aliphatic polymer based on dodecanedioic acid (DDA, 50%, 2) and ethylene glycol (EG, 50%, 1); PES2 (orange box) as a co-polymer with four different monomer units randomly distributed, namely, terephthalic acid (TPA, 37%, 3), isophthalic acid (IPA, 12%, 4), neopentyl glycol (NP, 48%, 6) and adipic acid (ADA, 3%, 5); PES3 (blue box, commonly indicated as PET) as an aromatic polymer based on terephthalic acid (TPA, 50%, 3) and ethylene glycol (EG, 50%, 1); and PES 4 (violet, dashed line box) as an aromatic polymer based on terephthalic acid (TPA, 50%, 3) and neopentyl glycol (NG., 50%, 6). These polyesters were also selected for their industrial relevance as part of the next generation of polymers.