The interfacial fracture toughness and the adhesion strength of two dissimilar materials are governed by the diffusion interfacial thickness and its mechanical characteristics. A new testing methodology is implemented here to estimate the actual interfacial thickness from a series of nanoindentations across the interface, under the same applied load, with tip radius and indentation depth many times larger than the interface thickness. The bimaterial system used is a semicrystalline polymer interface of isotactic polypropylene and linear low-density polyethylene. The laminate is prepared under a range of diffusion temperature to yield diffusion interfaces of 0 to 50 nm. A numerical relationship is developed using two-dimensional (2D) finite element simulation to correlate the true interfacial thickness, measured by transmission electron microscopy, with the experimentally estimated apparent interfacial thickness, derived from the transition domain of a series of indents across the interface. A range of material-pairs property combinations are examined for Young’s modulus ratio E1/E2 = 1 to 3, yield strength ratio σY1/σY2 = 1 to 2.5, and interfacial thickness of 0 to 100 nm. The proposed methodology and the numerically calibrated relationship are in good agreement with the true interfacial thickness.