Thermodynamic stability and unfolding kinetics of proteins are typically determined by monitoring protein unfolding with spectroscopic probes, such as circular dichroism (CD) and fluorescence. UV absorbance at 230nm (A 230 ) is also known to be sensitive to protein conformation. However, its feasibility for quantitative analysis of protein energetics has not been assessed. Here we evaluate A 230 as a structural probe to determine thermodynamic stability and unfolding kinetics of proteins. By using Escherichia coli maltose binding protein (MBP) and E. coli ribonuclease H (RNase H) as our model proteins, we monitored their unfolding in urea and guanidinium chloride with A 230 . Significant changes in A 230 were observed with both proteins on unfolding in the chemical denaturants. The global stabilities were successfully determined by measuring the change in A 230 in varying concentrations of denaturants. Also, unfolding kinetics was investigated by monitoring the change in A 230 under denaturing conditions. The results were quite consistent with those determined by CD. Unlike CD, A 230 allowed us to monitor protein unfolding in a 96-well microtiter plate with a UV plate reader. Our finding suggests that A 230 is a valid and convenient structural probe to determine thermodynamic stability and unfolding kinetics of proteins with many potential applications.