The ever-growing catalog of monomers being incorporated into block polymers affords exceptional control over phase behavior and nanoscale structure. The segregation strength, chi N, is the fundamental link between the molecular-level detail and the thermodynamics. However, predicting phase behavior mandates at least one experimental measurement of chi N for each pair of blocks. This typically requires access to the disordered state. We describe a method for estimating chi N from small-angle X-ray scattering measurements of the interfacial width between lamellar microdomains, t(x), in the microphase-separated melt. The segregation strength is determined by comparing t(x) to self-consistent field theory calculations of the intrinsic interfacial width, t(i), as a function of the mean-field chi N. The method is validated using a series of independent experimental measurements of t(x) and chi N, measured via the order-disorder transition temperature, T-ODT. The average absolute relative difference between chi N calculated from t(x) and the value calculated from T-ODT is a modest 11%. Corrections for nonplanarity of the interfaces are investigated but do not improve the agreement between the experiments and theory. Published 2019. This article is a U.S. Government work and is in the public domain in the USA. J. Polym. Sci., Part B: Polym. Phys. 2019 (c) 2019 Wiley Periodicals, Inc. J. Polym. Sci., Part B: Polym. Phys. 2019, 57, 932-940