Saffron, the most expensive spice in the world, is got by Crocus sativus L. stigmas. The production of this substance has attracted human interest, since ancient cultures, for its medicinal and culinary properties. Because of saffron high economic value, sometimes, since Middle Ages, it is adulterated with other vegetal materials, dyes or synthetic molecules. Object of this work was the study of one of the best world saffron: Civitaretenga (AQ, Central Italy) C. sativus. Taste, color and aroma of Civitaretenga spice were determined, according to international standards (ISO/Technical Specification 3632), to define its high quality. A biochemical approach was then applied to obtain a secondary metabolite profile of this product. So, crocins, total phenolic content, flavonoids and phenolic acids were detected by HPLC-DAD and spectrophotometric analysis. Moreover, in vitro antioxidant properties and in vivo antineoplastic effects, on highly metastatic murine B16-F10 melanoma cell line, were successfully revealed in Civitaretenga C. sativus extract. All these data confirmed the elevated quality of Civitaretenga saffron and its highly reducing and chemopreventive activity.