Interface stress is a surface thermodynamics quantity associated with the reversible work of elastically straining an internal solid interface. In a multilayered thin film, the combined effect of the interface stress of each interface results in an in-plane biaxial volume stress acting within the layers of the film that is inversely proportional to the bilayer thickness. We calculated the interface stress of an interface between {111} textured Ag and Ni on the basis of direct measurements of the dependence of the in-plane elastic strains on the bilayer thickness. The strains were obtained using transmission x-ray diffraction. Unlike previous studies of this type, we used freestanding films so that there was no need to correct for intrinsic stresses resulting from forces applied by the substrate that can lead to large uncertainties of the calculated interface stress value. Based on the lattice parameters of the bulk, pure elements, an interface stress of −2.02 ± 0.26 N/m was calculated using the x-ray diffraction results from films with bilayer thicknesses greater than 5 nm. This value is somewhat smaller than previous measurements obtained from as-deposited films supported by substrates. For smaller bilayer thicknesses the apparent interface stress becomes smaller in magnitude, possibly due to a loss of layering in the specimens.