In this paper, we characterized for the first time the painting materials in rock art panels of the Patagonian archaeological locality La Angostura using a methodological approach that combined micro-Raman spectroscopy, attenuated total reflection Fourier Transform infrared spectroscopy, and X-ray fluorescence spectroscopy. In this way, we obtained detailed information on the red, green, and black pigments as well as on accessory minerals present in the paints. Hematite was the chromophore in the red paintings while celadonite and glauconite were identified in the green motifs. A manganese oxide, presumably pyrolusite, was characterized as the black pigment. The pigment compositions were also compared to those of natural sediments collected along the valley of the Chubut River, but the components of the red and green sediment samples differed from those present in the rock art paints. In the red motifs of two of the rock art panels, a relevant finding was the presence of gypsum and anhydrite as a priming layer of the corrugated rock support onto which the red paints were applied. This revealed a different painting technique in comparison to that at the other analyzed panels of La Angostura where paints were applied as thin layers on a smooth surface of the rock support. As far as we know, this is the first report on the identification of gypsum and anhydrite as components of a preparation layer in Patagonian rock art. This result contributes to the open discussion on the origin and function of calcium sulfate in rock art.