Barley (Hordeum vulgaresubspp.vulgare) is the fourth most widely produced cereal today and is valuable for both animal and human consumption. Bacterial leaf streak (BLS) of barley is caused primarily byX. translucenspv. translucens (Xtt) and can lead to significant yield losses. There are currently no available control methods for BLS pathogens, but there are ongoing efforts to identify and characterize sources of resistance to Xtt in available barley germplasm. These screening projects require field trials which are time-consuming and challenged due to variables introduced by environmental and weather conditions, field conditions and other organisms that may be present. Reliable greenhouse phenotyping techniques are needed to accelerate screening of barley germplasm against more Xtt strains. In this study, we established a rapid greenhouse spray inoculation protocol for pathogen-plant phenotyping. This provides a framework for rapid germplasm screening before time-consuming and limited field nursery trials. Our method confirmed the moderate quantitative resistance phenotype discovered in field trials for the cultivars Quest and Tradition against the Xtt strain CIX95. One week old seedlings of the previously characterized resistant line PI329000 were not resistant relative to the susceptible check line MW14-5371-013. The cultivar Quest then demonstrated moderate quantitative resistance against a diverse panel of Xtt strains. Xtt subgroup did not influence BLS outcomes on tested barley lines. We found evidence to support the hypothesis that the virulence of modern strains is increasing, though this does not improve their ability to cause disease on Quest.