The current assays to confirm herbicide resistance can be time- and labor-intensive (dose–response) or require a skill set/technical equipment (genetic sequencing). Stakeholders could benefit from a rapid assay to confirm herbicide-resistant weeds to ensure sustainable crop production. Because protoporphyrinogen oxidase (PPO)-inhibiting herbicides rapidly interfere with chlorophyll production/integrity; we propose a new, rapid assay utilizing spectral reflectance to confirm resistance. Leaf disks were excised from two PPO-inhibiting herbicide-resistant (target-site [TSR] and non–target site [NTSR]) and herbicide-susceptible redroot pigweed (Amaranthus retroflexus L.) populations and placed into a 24-well plate containing different concentrations (0 to 10 mM) of fomesafen for 48 h. A multispectral sensor captured images from the red (668 nm), green (560 nm), blue (475 nm), and red edge (717 nm) wavebands after a 48-h incubation period. The green leaf index (GLI) was utilized to determine spectral reflectance ratios of the treated leaf disks. Clear differences of spectral reflectance were observed in the red edge waveband for all populations treated with the 10 mM concentration in the dose–response assays. Differences of spectral reflectance were observed for the NTSR population compared with the TSR and susceptible populations treated with the 10 mM concentration in the green waveband and the GLI in the dose–response assay. Leaf disks from the aforementioned A. retroflexus populations and two additional susceptible populations were subjected to a similar assay with the discriminating concentration (10 mM). Spectral reflectance was different between the PPO-inhibiting herbicide-resistant and herbicide-susceptible populations in the red, blue, and green wavebands. Spectral reflectance was not distinctive between the populations in the red edge waveband and the GLI. The results provide a basis for rapidly (∼48 h) detecting PPO-inhibiting herbicide-resistant A. retroflexus via spectral reflectance. Discrimination between TSR and NTSR populations was possible only in the dose–response assay, but the assay still has utility in distinguishing herbicide-resistant plants from herbicide-susceptible plants.