The production of indigo, primarily used by the denim industry, increases year by year, and is mainly of synthetic origin. The textile industry, on which its production depends, is responsible for 10% of greenhouse gases and 20% of water pollution. However, the source of this pigment/colorant, mainly based on petrochemistry, remains a key issue today. Extracting indigo from plants is becoming a popular answer and requires an understanding and evaluation of the entire process, from raw material to pigment recovery. In this study, the indigotin precursor, indoxyl, derived from the hydrolysis of O-glycosides biomass extracted in water, was oxidized to obtain the desired pigment. This step is the most sensitive, as variations have been observed during this phase. Consequently, the standardization of the oxidation process was established to determine the extract capacity to consistently produce the blue dye pigment. Partial hydrolysis of the O-glycosides, the indoxyl precursors, was identified as a factor causing this yield variability in the obtained extracts. Once the precursors were fully chemically hydrolyzed, plants harvested during summer and during a freezing period showed a similar capacity to produce indigotin, with values of 412 ± 25 ppm and 379 ± 0 ppm, respectively. This result showed that in freezing conditions, the enzymatic material was not available, resulting in the lack of indigotin formation. To address the use of oxidation in an alkaline medium, a spontaneous oxidation method was proposed. This method produced a purer indigotin pigment, with a 21.6% purity compared to 5.9% purity using air-mediated oxidation in an alkaline medium.