When glyphosate is applied to glyphosate-resistant (GR) crops, drift on to off-target sensitive crops may cause injury and mortality. An aerial application drift study was conducted in 2009 to determine biological effects of glyphosate on non-glyphosate-resistant (non-GR) cotton (Gossypium hirsutum L.) and non-GR soybean [Glycine max (L.) Merr.]. Glyphosate at 866 g ae/ha was applied using an Air Tractor 402B agricultural aircraft in an 18.3 m spray swath to crops at the two- to three-leaf stage. Visual plant injury, chlorophyll, shikimate, plant height, and shoot dry weight were determined at one, two, and three weeks after application (WAA) of glyphosate. Biological responses differed between crops as a function of downwind drift distance. For example, at 3 WAA soybean was dead 6 m downwind from the spray swath, whereas cotton sustained 85% visual injury. Plant injury was not observed beyond 25.6 m downwind in soybean and 35.4 m downwind in cotton at 3 WAA. Chlorophyll reduction was higher (80%) in soybean compared with cotton (43%) at 0 m from the edge of the spray at 1 WAA. Shikimate levels 1 WAA decreased from 1518% at 0 m to 209% at 35.4 m downwind in soybean; at the same sampling time shikimate levels in cotton decreased from 464% at 0 m to 0% at 35.4 m. At 35.4 m downwind, shoot dry weight (5–13%) and plant height (6–8%) were reduced in both crops at 3 WAA. The biological response of soybean and cotton to glyphosate drift decreased with increased distance from the edge of spray swath. These biological data suggested that soybean was more susceptible to glyphosate drift than cotton and elevated shikimate level could be used as a sensitive indicator to confirm plant exposure to glyphosate drift.