Early detection of grape phylloxera (Daktulosphaira vitifoliae) infestation is vital for the implementation of post-outbreak quarantine in Australia. Remote sensing systems exploit changes in leaf pigment content associated with plant stress and offer a real possibility of a phylloxera-specific detection system. Pre-visual, symptomatic changes in the pigment content of phylloxera-infested grapevine leaves were investigated using high performance liquid chromatography (HPLC) as a potential aid to improve current phylloxera detection methods. A glasshouse trial was established to characterize the response of two grapevine varieties, Vitis vinifera ‘Cabernet Sauvignon’ and ‘Shiraz’, to phylloxera infestation, in a controlled environment. Field trials were conducted on two grapevine varieties, V. vinifera ‘Cabernet Sauvignon’ and ‘Pinot Noir’, at two sites, to compare grapevine response to phylloxera infestation under field conditions. A reduction in the leaf chlorophyll content and an increase in photoprotective pigment concentrations were observed in leaves of phylloxera-infested grapevines compared to uninfested vines. With further investigation, the identification of grapevine leaf pigment responses to phylloxera infestation may prove useful for the rapid, non-invasive, detection of phylloxera in commercial vineyards.