Antarctic palaeoclimate evolution and vegetation history after the formation of a continent-scale cryosphere at the Eocene/Oligocene boundary, 33.9 million years ago, has remained a matter of controversy. In particular, the reconstruction of terrestrial climate and vegetation has been strongly hampered by uncertainties in unambiguously identifying in situ as opposed to reworked sporomorphs that have been transported into Antarctic marine sedimentary records by waxing and waning ice sheets. Whereas reworked sporomorph grains over longer non-successive geological time scales are easily identifiable within younger sporomorph assemblages (e.g., Permian sporomorphs in Pliocene sediments), distinguishing in situ from reworked material in palynological assemblages over successive geological time periods (e.g., Eocene sporomorphs in Oligocene sediments) has remained problematic. This study presents a new quantitative approach to identifying in situ grains from a marine sediment core from circum-Antarctic waters. We measured the fluorescence signature and mean red, green and blue, brightness, intensity and saturation values of selected pollen and spore taxa from Eocene, Oligocene and Miocene sediments from the Wilkes Land margin Site U1356 (East Antarctica) recovered during Integrated Ocean Drilling Program (IODP) Expedition 318. Our study identified statistically significant differences in mean red fluorescence values of in situ sporomorph taxa against age. We conclude that red fluorescence is a reliable parameter to identify the presence of in situ pollen and spores in Antarctic marine sediment records from the circum-Antarctic realm that are influenced by glaciation and extensive reworking. Our study provides an essential new tool required to accurately reconstruct Cenozoic terrestrial climate change on Antarctica using fossil pollen and spores.