Non-destructive analysis of indigo dye in biological and textile samples is presented. Using different but complementary techniques of vibrational spectroscopy (FT-Raman and ATR-IR) detection of indigo in various samples is performed. This work shows the possibility of both vibrational analytical methods to identify indigo directly in plant tissue, commercially available pigments and cotton and silk textiles. The experimental work is supported by quantum-chemical calculations at the B3LYP/6-311++g(d,p) level of theory. Additionally, in order to follow the process of indigo formation upon leaf drying Raman mapping of Polygonum tinctorium is demonstrated.