An analytical strategy, based on the development of two HPLC methods with spectrophotometric (UV), spectrofluorimetric (FL) and mass spectrometric (MS) detection, has been developed to investigate the presence and to quantitate two important chemopreventative coumarins, auraptene and umbelliferone, in foodstuffs. The analytes were determined in fruits, and fruit parts, of plants belonging to the Citrus, Poncirus and Fortunella genera, to test their nutraceutical potential. The method validation has been carried out according to international guidelines, with good results in terms of precision (RSD < 6.9%) and extraction yields (> 91%). Application to the quantitative analysis of auraptene and umbelliferone in several kinds of Citrus fruits was successful, providing reliable and consistent data. Exploiting three different kinds of detection, the analytical methodology proposed herein has demonstrated to be sound but versatile, as well as reliable. Performances and results were compared and always found in good agreement among themselves. Thus, this approach is suitable for the identification and the simultaneous quantitation of auraptene and umbelliferone in Citrus fruits, with the aim of evaluating their nutraceutical potential.