This study shows that protein size, folding and function can be characterized simultaneously on a single instrument using nanoliter sample volumes. The authors combined Taylor dispersion analysis (TDA), Flow Induced Dispersion Analysis (FIDA) and in-line intrinsic fluorescence to follow urea-induced unfolding of human serum albumin (HSA). FIDA measured the binding affinity between HSA and the small-molecule ligand fluorescein, showing binding weakened from a dissociation constant of 25.1 µM at 0 M urea and was fully lost at 4 M urea. In parallel, TDA reported the growth in hydrodynamic radius from 4.0 to 5.7 nm, and intrinsic fluorescence probed local changes near tryptophan and tyrosine residues. Together these readouts gave a detailed, automated picture of denaturation, useful for biophysics and stability screening of protein drug candidates.