Objective The mechanical strength of the cervix relies on the cross-linking of the tissue's collagen network. Clinically, the internal os is functionally distinct from the external os. We sought to detect specific collagen cross-links in human cervical tissue and determine whether cross-link profiles were similar at the internal and external os. Study Design Transverse slices of cervical tissue were obtained at the internal and external os from 13 nonpregnant, premenopausal women undergoing a benign hysterectomy. To understand how cross-links were distributed throughout the entire cervix and at the internal and external os, biopsies were obtained from 3 circumferential zones in 4 quadrants from each slice. Biopsies were pulverized, lyophilized, reduced with sodium borohydride, hydrolyzed with hydrochloric acid, and reconstituted in heptafluorobutyric acid buffer. Hydroxyproline was measured by ultraperformance liquid chromatography-electrospray ionization tandem mass spectrometry (UPLC-ESI-MS/MS), converted to total collagen, and normalized by dry weight. Collagen cross-links pyridinoline (PYD), deoxypyridinoline (DPD), dihydroxylysinonorleucine (DHLNL), and the nonenzymatic advanced glycation end product pentosidine (PEN) were measured by UPLC-ESI-MS/MS and reported as cross-link density ratio (cross-link/total collagen). Generalized estimated equation analysis was used to compare results between the internal and external os and to compare quadrants and zones within slices from the internal and external os to determine if cross-link profiles were similar. Results A total of 592 samples from 13 patients were analyzed. Collagen cross-links are detectable in the human cervix by UPLC-ESI-MS/MS. When comparing all samples from the internal and external os, similar levels of collagen content, PYD, DHLNL, and DPD were found, but PEN density was higher at the external os (0.005 vs 0.004, P = .001). When comparing all internal os samples, significant heterogeneity was found in collagen content and cross-link densities across zones and quadrants. The external os exhibited heterogeneity only across zones. Conclusion Collagen cross-links (PYD, DPD, DHLNL, and PEN) are detectable by UPLC-ESI-MS/MS in the human cervix. The internal os exhibits significant collagen cross-link heterogeneity compared with the external os. Further studies are needed to evaluate how collagen cross-link heterogeneity correlates to the mechanical strength and function of the human cervix.