This study demonstrated that Flow Induced Dispersion Analysis (FIDA) can characterize weak host–guest binding interactions between cyclodextrins and small-molecule guests. The authors synthesized a fluorescein-labeled L-tryptophan probe and used FIDA to measure changes in the probe's hydrodynamic radius as it associated with α-, β- and γ-cyclodextrins, running both direct binding assays and competitive displacement assays with unlabeled tryptophan. FIDA was used to quantify complex formation and derive apparent binding affinities, with β-cyclodextrin showing the strongest interaction (apparent association constant K ≈ 470 M⁻¹) and α- and γ-cyclodextrins binding more weakly. The work establishes FIDA as a solution-based, immobilization-free approach for probing low-affinity supramolecular interactions relevant to drug delivery and molecular sensing.