The stability of proteins is tuned by evolution to enable them to perform their cellular functions for the success of an organism. Yet, most of the arsenal of biophysical techniques at our disposal to characterize the thermodynamic stability of proteins is limited to in vitro samples. We describe an approach that we have developed to observe a protein directly in a cell and to monitor a fluorescence signal that reports the unfolding transition of the protein, yielding quantitatively interpretable stability data in vivo. The method is based on incorporation of structurally nonperturbing, specific binding motifs for a bis-arsenical fluorescein derivative in sites that result in dye fluorescence differences between the folded and unfolded states of the protein under study. This fluorescence labeling approach makes possible the determination of thermodynamic stability by direct urea titration in Escherichia coli cells. The specific case study we describe was carried out on the predominantly β-sheet intracellular lipid-binding protein, cellular retinoic acid-binding protein (CRABP), expressed in E. coli.