The effect of annealing temperature on molecular interactions at the interface of polymer laminates is reported. Depth profiling has been carried out by using confocal Raman microspectroscopy to study poly(acrylonitrile)/poly(vinyl alcohol) (PAN/PVOH) and poly(acrylonitrile)/poly(acrylic acid) (PAN/PAA) laminates. The laminates have been annealed at 65, 75, and 90 °C. It is demonstrated that the degree of hydrogen-bonding interaction between the nitrile and alcohol groups near the interfacial region changes between laminates annealed at different temperatures. Increasing the annealing temperature up to near the glass transition temperature, Tg, of both polymers facilitates hydrogen bonding. However, above Tg, as a result of molecular flexibility, weakening of such interactions begins. Due to the lower molecular weight of PAA, and thus greater mobility in comparison with PVOH, the interfacial region of a PAN/PAA laminate is broader than that of a PAN/PVOH laminate. Hydrogen-bonding interaction between PAN and PAA was not observed. This result is rationalized by the hypothesis that, as a result of intramolecular interaction between the carbonyl and hydroxyl groups of PAA, formation of intermolecular hydrogen bonding has been hindered. In addition, the variations in the full width at half-height (FWHH) of the v(C≡N) band of a PAN–PVOH blend has been mapped by using the same technique.