A new method is based on the flow injection gradient equipped with an acousto-optic tunable filter (AOTF) detector has been developed for the determination of binding constants of substrates with cyclodextrins or micelles. The concentration gradient produced by this method eliminates the need to prepare series of mixtures in which the concentrations of one of the reactants are varied. The fast scanning ability of the AOTF enables the detector to record the whole absorption spectrum of the mixture as it flows through the flow cell. Binding constants can be rapidly determined. The effectiveness of the method was evaluated by determining binding constants of inclusion complex formation between beta-cyclodextrin (beta-CD) and phenolphthalein and of the incorporation of organic molecules (e.g., p-toluenesulfonic acid (PTS) and benzophenone (BP)) into micelles (CTAB) under steady state condition (no flow) and compared with values determined under the flow injection gradient. The method is particularly effective for charged substrates, e.g., the binding constants obtained between PTS and CTAB and between phenolphthalein and beta-CD. Neutral substrate, BP, gives relatively poor accuracy and reproducibility, and these may be due to the adsorption of the substrate onto the flow device.