AbstractMaya blue is a hybrid pigment where an organic component, indigo, is incorporated in a porous clay. Despite its widespread use in the Mesoamerican artistic production and numerous studies devoted to understand the type of interactions between indigo and the host framework, its technology has not been completely unravelled yet. In this study portable non-invasive UV–vis reflection spectroscopy is proposed as a robust method for in situ investigation of Maya blue pigments. Laboratory mock-ups of powder Maya blue have been prepared employing different synthesis procedures (varying the nature of: clays, indigo–clay ratios, heating temperatures and time). The goodness of the prepared Maya blue samples—namely the occurrence of indigo–clay interactions—have been probed by micro-Raman spectroscopy and related UV–vis spectral markers have been identified. DFT calculations as well have been performed to deeply explain UV–vis profiles. The set of spectral markers have been finally exploited to interpret spectra recorded on Mesoamerican pictorial codices, through a multi-technique approach based on exploring the UV–Vis properties of the blue paint supported by the FT-IR vibrational study of the inorganic clays. The characterization of blue colours on pre-Hispanic and colonial Mesoamerican codices contribute to a better understanding of the compositional variability of these painting materials and to point out the existence of different technological traditions of colour preparation in ancient Mesoamerica.