Biotic stresses such as fungal pathogens significantly affect global crop yields. Understanding of the plant-pathogen interactions during root infection, especially in monocot crops, remains limited compared to fungal colonizations of dicots. The infection process of several cereal crop root-damaging fungi and oomycetes is highly similar to root infections by the pathogen model Phytophthora palmivora. Here, we describe a protocol for quantifying root infection of Phytophthora palmivora in barley or related monocots using reverse transcriptase quantitative PCR. This method can be used to produce relative expression values for the oomycete within roots based on RNA or DNA. Different mutations in the host can, therefore, be quantitatively assessed for their impact on infection. This allows us to identify plant processes underpinning susceptibility to filamentous fungal colonization within monocot models and thus can facilitate the development of possible resistance strategies.