Fluorescent dyes, for example, 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid dipotassium salt (bis-ANS) are extensively used to detect nonnative protein structures in therapeutic protein products, for example, during formulation development by monitoring the greatly enhanced dye fluorescence upon binding to nonnative species. Our aim was to characterize the level of heterogeneity of bis-ANS binding sites in a thermally stressed monoclonal antibody (IgG) formulation by steady-state fluorescence, time-resolved fluorescence and isothermal titration calorimetry (ITC), and to obtain apparent dissociation constants (Kd ) by data fitting. Because the methods differ in their underlying measurement principles, they provide different information on binding properties of bis-ANS to thermally stressed IgG. We found very heterogeneous bis-ANS binding sites on thermally stressed IgG, with apparent Kd values ranging from as low as 50 nM (time-resolved fluorescence) to 63 μM (ITC). Steady-state fluorescence and ITC gave insight into an overall binding affinity of a wide population of dye binding sites with micromolar Kd values. Time-resolved fluorescence was particularly sensitive to high-affinity binding sites with nanomolar Kd s. The heterogeneity of the bis-ANS binding sites reflects the complex, heterogeneous nature of the heat-stressed IgG used in this study. To probe such heterogeneity adequately, one should apply complementary analytical methods under various experimental conditions as presented in this paper. © 2010 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 100:1294-1305, 2011.