Human retinal macular pigment (MP) is formed by the carotenoids lutein and zeaxanthin (including the isomer meso-zeaxanthin). MP has several functions in improving visual performance and protecting against the damaging effects of light, and MP levels are used as a proxy for macular health-specifically, to predict the likelihood of developing age-related macular degeneration. While the roles of these carotenoids in retinal health have been the object of intense study in recent years, precise mechanistic details of their protective action remain elusive. We have measured the Raman signals originating from MP carotenoids in ex vivo human retinal tissue, in order to assess their structure and conformation. We show that it is possible to distinguish between lutein and zeaxanthin, by their excitation profile (related to their absorption spectra) and the position of their ν1 Raman mode. In addition, analysis of the ν4 Raman band indicates that these carotenoids are present in a specific, constrained conformation in situ, consistent with their binding to specific proteins as postulated in the literature. We discuss how these conclusions relate to the function of these pigments in macular protection. We also address the possibilities for a more accurate, consistent measurement of MP levels by Raman spectroscopy.