The aim of the study was to evaluate the effect of various fertilization treatments, including nitrogen, potassium and phosphorus fertilization, in long-term experiments for selected UAV (unmanned aerial vehicle)-derived spectral vegetation indices (NDVI—Normalized Difference Vegetation Index, NDRE—Normalized Difference Red Edge Index, VARI—Visible Atmospherically Resistant Index, TGI—Triangular Greenness Index, SIPI2—Structure Insensitive Pigment Index 2, LCI—Leaf Chlorophyll Index, BNDVI—Blue Normalized Difference Vegetation Index, GNDVI—Green Normalized Difference Vegetation Index, MCARI—Modified Chlorophyll Absorption in Reflective Index) based on multispectral (bands in the range of visible light and near infra-red) images of winter rye. The strongest effect on the studied vegetation indices was nitrogen fertilization, which discriminated values of most of the vegetation indices. The effect of phosphorus and potassium fertilization on the studied vegetation indices was much weaker. The treatments with nitrogen fertilization had significantly higher values of most vegetation indices in comparison to treatments without nitrogen. This was confirmed by principal component analysis (PCA), in which treatments without nitrogen fertilization were very different in comparison to all other treatments where nitrogen fertilization was applied. The effect of phosphorus and potassium fertilization on most of vegetation indices was relatively weak and not significant in most experiments. Only for rye cultivated in monoculture was the effect of phosphorus fertilization significant for most of vegetation indices in early growth stages. In later growth stages (heading and flowering) the effect of phosphorus fertilization was significant in rye monoculture for the SIPI2 vegetation index. Mean SIPI2 was higher for the fertilization treatment CaNPK in comparison to CaKN (without P fertilization). The effect of potassium fertilization on the studied vegetation indices was very weak, and in most cases not significant. The effect of nitrogen fertilization on vegetation indices was much stronger than effect of both potassium and phosphorus fertilization.