The ever‐growing catalog of monomers being incorporated into block polymers affords exceptional control over phase behavior and nanoscale structure. The segregation strength, χN, is the fundamental link between the molecular‐level detail and the thermodynamics. However, predicting phase behavior mandates at least one experimental measurement of χN for each pair of blocks. This typically requires access to the disordered state. We describe a method for estimating χN 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 χN. The method is validated using a series of independent experimental measurements of t_x and χN, measured via the order–disorder transition temperature, T_ODT. The average absolute relative difference between χN 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 © 2019 Wiley Periodicals, Inc. J. Polym. Sci., Part B: Polym. Phys. 2019, 57, 932–940