<p>Endometriosis is a gynecological condition characterized by the uncontrolled growth of endometrial-like tissue outside the uterine cavity. Although highly prevalent, the biological mechanisms of endometriosis are poorly understood, and the disease is often misdiagnosed due to the unavailability of pre-operative diagnostic methods. Thus, better characterization of molecular markers of endometriosis is critical to improve our understanding of the disease and management of patients. Here, we used matrix-assisted laser desorption ionization mass spectrometry (MALDI MS) imaging to investigate protein and N-glycan molecular signatures in eutopic endometrium and endometriosis lesions from tissues prospectively collected from patients. MALDI MS imaging of intact proteins revealed neutrophil defensins detected at higher relative abundances in regions of endometriosis lesions, providing evidence that immune/inflammatory processes may be associated with endometriosis. Various alterations in N-glycan molecular profiles were also observed when comparing endometriosis lesions and eutopic endometrium, including a significantly higher detection of fucosylated N-glycans and increased levels of branching from biantennary to tri- and tetra-antennary structures in endometriosis lesions. These results provide evidence that alterations in N-glycosylation machinery are involved in dysregulation of cellular and signaling processes and may contribute to endometriosis development. A co-localization analysis identified several tryptic peptides detected within regions of endometriosis lesions, suggesting extracellular matrix (ECM) proteins such as collagen may contribute to the development of endometriosis lesions. Overall, by characterizing molecular alterations in proteins and N-glycans, the results of this study provide novel insights into potential mechanisms involved in endometriosis pathogenesis.</p> Graphical Abstract <p></p>