The study and characterization of organic dyes in textiles, wastewaters, food, cosmetics and forensic specimens is an increasingly important field of research and a challenge for analytical chemistry. Sensitive and specific methods are required to determine molecular markers at very low concentrations and to detect and identify their relative degradation products. In this field, the present paper deals with the development of an extremely sensitive analytical strategy able to identify synthetic organic dyes even after ageing, such as photo-oxidation resulting in severe fading, by identifying specific stable markers. Rhodamine B (C.I. 45,170), belonging to the class of xanthenes, was chosen as representative of fugitive dyes of high commercial and historical importance, in the frame of a research promoted by the European platform IPERION-CH. The behaviour of Rhodamine B used as a dyestuff for wool yarns, subjected to accelerated photo-ageing, was investigated by chromatographic, spectroscopic and high resolution mass spectrometric techniques. The ultra-sensitive analytical method developed, based on core shell reverse phase analytical column separation, allowed us to characterize Rhodamine B oxidation products investigating their mass fragmentation and rationalizing the resulting ageing pathway. These degradation compounds, characterized in artificially aged reference wool yarns, confirmed to be valuable markers for the identification of Rhodamine B even in a complex historical textile matrix as a red Florentine velvet cope subjected to restoration in the 20th century.