Nanoindentation combined with atomic force microscopy was applied to measure the fracture toughness of polystyrene/glass interfaces. Film delamination occurs when the inelastic penetration depth approximately equals or exceeds the film thickness. The delamination size was accurately measured using atomic force microscopy. Using multilayer indentation and annular-plate analyses, the interfacial fracture toughness was then assessed. The values obtained from the two analyses are in good agreement with the fracture toughness of the interface being approximately 350 mJ/m2. By appropriate fracture surface characterization, it was shown that fracture occurs along the polystyrene/glass interface. Crack arrest marks were observed, and their possible cause discussed. On the basis of the morphology of the fracture surface, the fracture toughness was also evaluated using a process zone analysis. The result agrees well with those obtained from the other two analyses.