Biosurfactants are produced by microorganisms and represent amphiphilic compounds with polar and non-polar moieties; hence they can be used to stabilize emulsions, e.g., in the cosmetic and food sectors. Their structure and its changes when exposed to light and elevated temperature are yet to be fully understood. In this study, we demonstrate that attenuated total reflection infrared (ATR-IR) spectroscopy is a useful tool for the analysis of biosurfactants, using rhamnolipids produced by fermentation as an example. A key feature is that the analytical method does not require sample preparation despite the high viscosity of the purified natural product.