Agrobacterium-mediated transient expression (AMTE) has been widely used for high-throughput assays of gene function in diverse plant species. However, its application in monocots is still limited due to low expression efficiency. Here, by using histochemical staining and a quantitative fluorescence assay of β-glucuronidase (GUS) gene expression, we investigated factors affecting the efficiency of AMTE on intact barley plants. We found prominent variation in GUS expression levels across diverse vectors commonly used for stable transformation and that the vector pCBEP produced the highest expression. Additionally, concurrent treatments of plants with one day of high humidity and two days of darkness following agro-infiltration also significantly increased GUS expression efficiency. We thus established an optimized method for efficient AMTE on barley and further demonstrated its efficiency on wheat and rice plants. We showed that this approach could produce enough proteins suitable for split-luciferase assays of protein-protein interactions on barley leaves. Moreover, we incorporated the AMTE protocol into the functional dissection of a complex biological process such as plant disease. Based on our previous research, we used the pCBEP vector to construct a full-length cDNA library of genes upregulated during the early stage of rice blast disease. A subsequent screen of the library by AMTE identified 15 candidate genes (out of ~2000 clones) promoting blast disease on barley plants. Four identified genes encode chloroplast-related proteins: OsNYC3, OsNUDX21, OsMRS2-9, and OsAk2. These genes were induced during rice blast disease; however, constitutive overexpression of these genes conferred enhanced disease susceptibility to Colletotrichum higginsianum in Arabidopsis. These observations highlight the power of the optimized AMTE approach on monocots as an effective tool for facilitating functional assays of genes mediating complex processes such as plant-microbe interactions.