Monitoring protein size changes has versatile applications in studying protein folding/unfolding, conformational rearrangements, and ligand binding. Traditionally, FRET has been used to obtain this information. However, the use of FRET often requires covalent attachment of exogenous fluorophores. Although intrinsic FRET also exists between tyrosine and tryptophan residues, it has been underused because of tyrosinate formation and spectroscopic overlap. Herein, we clarified the concern of tyrosinate formation and mathematically deconvoluted tyrosine/tryptophan fluorescence spectra. We define a new parameter called FirbY-W (fluorescence intensity ratio between tyrosine and tryptophan) to reflect protein sizes. We demonstrate its applications in studying protein unfolding using several model proteins. In all the cases, our method offers superior sensitivity, data quality, and robustness compared with traditional techniques. The unique power of our method is in its ability to detect elusive conformational changes of intrinsically disordered proteins (IDP). The lack of structure makes IDPs unsuitable for CD or tryptophan fluorescence characterization. Using histone mRNA stem-loop binding protein (SLBP) as an example of disordered proteins, we showed that our method is capable of detecting conformational changes caused by phosphorylation, which are effectively invisible for traditional spectroscopic methods. Our method can also be used to detect RNA binding of disordered proteins.