Technical Abstract: Plant cells possess a number of membrane bound organelles that play important roles in compartmentalizing a large number of biochemical pathways and physiological functions that have potentially harmful intermediates or by-products. The plasma membrane is particularly important as it holds the entire cellular structure whole and is at the interface between the cell and its environment. Consequently, breaches in the integrity of the lipid bilayer, often via reactive oxygen species (ROS)-induced stress membrane peroxidation, result in uncontrolled electrolyte leakage and in cell death. A simple 3-step bioassay was developed to identify compounds that cause electrolyte leakage and to differentiate light-dependent mechanisms of action from those that work in darkness. Herbicides representative of all known modes of action (as well as several natural phytotoxins) were selected to survey their effects on membrane integrity of cucumber cotyledon discs. The most active compounds were those that are known to generate ROS such as the electron diverters and uncouplers (paraquat and dinoterb) and those that either were photodynamic (cercosporin) or caused the accumulation of photodynamic products (acifluorfen-methyl and sulfentrazone). Other active compounds targeted lipids (diclofop-methyl, triclosan and pelargonic acid) or formed pores in the plasma membrane (syringomycin). Herbicides that inhibit amino acid, protein, nucleotide, cell wall or microtubule synthesis did not have any effect. Therefore, it was concluded that the plant plasma membrane is a good biomarker to help identify certain herbicide modes of action and their dependence on light for bioactivity