Turfgrass managers currently have few readily available means of evaluating herbicide resistance in annual bluegrass during the growing season. Research was conducted to determine if agar-based diagnostic tests developed for agronomic weeds could be used to reliably confirm herbicide resistance in annual bluegrass harvested from golf course turf. Annual bluegrass phenotypes with target-site resistance to acetolactate synthase (ALS; R3, R7), enolpyruvylshikimate-3-phosphate synthase (EPSPS; R5), and photosystem II (PSII; R3, R4) inhibiting herbicides were included in experiments along with an herbicidal susceptible phenotype (S). Single tiller plants were washed free of soil and transplanted into autoclavable polycarbonate plant culture boxes filled with plant tissue culture agar amended with a murashigee-skoog medium and trifloxysulfuron (6.25, 12.5, 25, 50, 75, 100, or 150 μM), glyphosate (0, 6, 12, 25, 50, 100, 200, or 400 μM), or simazine (0, 6, 12, 25, 50, 100, 200, or 400 μM). Mortality in agar was assessed 7 to 10 days after treatment (depending on herbicide) and compared to responses observed after treating individual plants of each phenotype with trifloxysulfuron (28 g ai ha-1), glyphosate (1120 g ae ha-1), or simazine (1120 g ai ha-1) in an enclosed spray chamber. Fisher’s exact test (α = 0.05) determined that mortality in agar with 12.5 μM trifloxysulfuron and 100 μM glyphosate was not significantly different than treating whole plants via traditional spray application. Mortality with all concentrations of simazine in agar was significantly different than that observed after treating resistant and susceptible phenotypes via traditional spray application. Our findings indicate that an agar-based diagnostic assay can be used to detect annual bluegrass resistance to ALS- or EPSPS-inhibiting herbicides in less than 10 days; however, additional research is needed to refine this assay for use with PSII-inhibiting herbicides.