In this work, we present a methodology for the determination of the depth of demineralization in dental enamel caused by extended use of an Over-The-Counter (OTC) whitening product. Teeth whitening is a very common practice in Dentistry, but concerns have been raised regarding the invasiveness of the treatment, especially regarding OTC products, that can be used without medical supervision and sometimes with concentrations of active agent that exceed the allowed regulations. In this work, we studied tooth enamel samples, treated with a whitening product during an extended period of time, both directly on the enamel surface and in the cross-section. Specimens were analyzed using microbeam X-Ray Fluorescence (micro-XRF) using polycapillary optics to obtain a spot down to 25 μm. Due to the relatively large spot size of our setup point analysis of the cross-sections would be inadequate. This way, line scans were performed instead, before and after whitening, and using appropriate data treatment the depth of demineralization was inferred. The used methodology indicated an average demineralization depth of 25 μm, the same order of magnitude as the aprismatic enamel layer.