Optimum herbicide use is a key factor affecting the success of any integrated weed management strategy. The main objective of the current study was to implement a method based on spectrometer measurements for the in situ evaluation of herbicide efficacy and the detection of potentially herbicide-resistant weeds. Field trials were conducted in Greece between 2018 and 2020 in several durum wheat fields (Triticum durum Desf.). In all trials, the overall effect of herbicide application on the recorded Normalized Difference Vegetation Index (NDVI) values (at 1 and 2 wk after treatment [WAT]) was significant (P ≤ 0.001). For the majority of the surveyed fields, low NDVI values were recorded after 2,4-D application and a mixture of clopyralid + florasulam from 1 WAT, suggesting their increased efficacy. In several cases, the application of pyroxsulam + florasulam resulted in significant NDVI reductions at 2 WAT. As observed at the end of the growing seasons, the herbicides that reduced NDVI resulted in lower weed biomass. Strong correlations were observed between weed aboveground biomass and NDVI (2 WAT). In particular, R² values were 0.8234 to 0.8649, 0.8453, 0.8595, 0.8149, and 0.8925 for the Aliartos, Thiva, Domokos, Larissa, and Orestiada fields, respectively. The overall effects of herbicide application on wheat grain yield were also significant (P ≤ 0.001). Pot experiments confirmed that the high NDVI values in some cases could be attributed to the presence of herbicide-resistant weeds. For instance, the resistance indices of two accessions of catchweed bedstraw (Galium aparine L.) to mesosulfuron-methyl + iodosulfuron-methyl-sodium ranged between 9.7 and 13.2, whereas one sterile oat [Avena sterilis L. ssp. ludoviciana (Durieu) Gillet & Magne] accession was 8.8 times more resistant to fenoxaprop-p-ethyl than a susceptible one. The present study is targeted at making a significant contribution toward establishing cause–effect relationships and presenting a useful tool for developing more effective weed management practices in more arable crops and under different soil and climatic conditions.