Microsamples of pink cosmetic powders from the Greco–Roman period were analyzed using two complementary analytical approaches for identification of the colouring agents (lake pigments originally manufactured from madder plants with an inert binder, usually a metallic salt) present in the samples. The first technique was a methanolic acidic extraction of the archaeological samples with an additional ethyl acetate extraction of the anthraquinone-type colouring agents which were identified using high performance liquid chromatography coupled to electrospray ionization with high resolution mass spectrometry (LC–ESI–HRMS), and the second was direct analysis of a microsample by laser desorption ionization–mass spectrometry (LDI–MS). The latter technique is well suited when the quantity of samples is very low. This soft ionization technique enables the detection of very small quantities of compounds using the combination of positive and negative-ion modes. It was also successfully applied for the direct analysis of some laboratory-made reference compounds. However, the presence of lead in one of these ancient samples induced a spectral suppression phenomenon. In this case and conditional on a sufficient quantity of available sample, the former method is better adapted for the characterization of these anthraquinone-type molecules. This study also confirmed that purpurin, munjistin, and pseudopurpurin are the principal colouring agents present in these ancient cosmetic powders constituted from madder plants.