A novel kinetic spectrophotometic method for the determination of Tween 80 based on its interaction with 5(p-dimethylaminobenzylidene)rhodanine (PDR) in alkaline media is reported. The effect of variable on the rate of interaction of Tween 80 and PDR was investigated in order to establish the optimum conditions. The interaction was monitored spectrophotometically and change in absorbance (DeltaA) of PDR at 464nm at times of 30 and 270s was used as an analytical parameter. Tween 80 can be measured in the range of 2.5x10(-5) to 1.25x10(-3)M with detection limit of 1.5x10(-5)M. The relative standard deviation for eight replicate determinations of 2x10(-4) and 1x10(-3)M of Tween 80 solution was 4.08 and 3.88%, respectively. This method was used to determine Tween 80 in biscuit and multivitamin syrup.