This laboratory experiment tested the ability of the spectral index called ‘area under curve normalised to maximal band depth’ (ANMB) to track dynamic changes in the xanthophyll cycle of Norway spruce (Picea abies (L.) Karsten) needles. Four-year-old spruce seedlings were gradually acclimated to different photosynthetic photon flux densities (PPFDs) and air temperature regimes. The measurements were conducted at the end of each acclimation period lasting for 11 days. A significant decline in the chlorophylls to carotenoids ratio and the increase of the amount of xanthophyll cycle pigments indicated a higher need for carotenoid-mediated photoprotection in spruce leaves acclimated to high PPFD conditions. Similarly, the photochemical reflectance index (PRI) changed from positive to negative values after changing light conditions from low to high intensity as a consequence of the increase in carotenoid content. Systematic responses of PRI to the de-epoxidation state of xanthophyll cycle pigments (DEPS) were, however, observed only during high temperature treatments and after the exposition of needles to high irradiance. The ANMB index computed from needle reflectance between 507 and 556 nm was able to track dynamic changes in DEPS without any influence induced by changing the content of leaf photosynthetic pigments (chlorophylls, carotenoids).