High-intensity focused ultrasound (HIFU) is often used to create lesions, or regions of tissue destruction due to heating and cavitation activity, most often in tumors or other diseased tissues. However, the acoustic properties of tissues denatured by heat are not very different from those of untreated tissue, making lesion detection and quantification difficult by ultrasound alone. Autofluorescence refers to the broadband emission of light within materials that are stimulated by narrowband incident light, typically from a laser. It is a common characteristic of lipids, proteins, and other biomolecules, and the “autofluorescence spectrum” is a function of the state of the material. We have examined irreversible shifts in the dominant autofluorescence spectra of proteins denatured by HIFU heating. These shifts appear to be related to protein conformational changes due to denaturation. We report on the feasibility of using optical autofluorescence as a means of quantifying in vitro lesion formation by HIFU for optically accessible tissues.