Sensor technologies are expedient tools for precision agriculture, aiming for yield protection while reducing operating costs. A portable sensor based on chlorophyll fluorescence imaging was used in greenhouse experiments to investigate the response of sugar beet and soybean cultivars to the application of herbicides. The sensor measured the maximum quantum efficacy yield in photosystem II (PS-II) (Fv/Fm). In sugar beet, the average Fv/Fm of 9 different cultivars 1 d after treatment of desmedipham plus phenmedipham plus ethofumesate plus lenacil was reduced by 56% compared to the nontreated control. In soybean, the application of metribuzin plus clomazone reduced Fv/Fm by 35% 9 d after application in 7 different cultivars. Sugar beets recovered within few days from herbicide stress while maximum quantum efficacy yield in PS-II of soybean cultivars was reduced up to 28 d. At the end of the experiment, approximately 30 d after treatment, biomass was reduced up to 77% in sugar beet and 92% in soybean. Chlorophyll fluorescence imaging is a useful diagnostic tool to quantify phytotoxicity of herbicides on crop cultivars directly after herbicide application, but does not correlate with biomass reduction.