Host-induced gene silencing (HIGS) and spray-induced gene silencing (SIGS) have been shown to be effective in controlling plant pathogens. Understanding the role of both natural and engineered trans-kingdom RNAi in plant immunity is recognized as important for reducing FHB. F.g. grows within the apoplast prior to infecting cells so understand that growth environment is critical to developing a better understanding of the plant-pathogen interaction. Previous work in the lab has shown that exosomes isolated from barley apoplastic fluid from F.g. infected plants contain increased amounts of small heat shock proteins, annexins, GAPDH and GSH enzymes, among other induced proteins. To better understand the apoplast proteome in response to F.g. infection, a time-course was set up using the barley variety Conlon and leaf tissue was sampled at: 0, 24, 48, and 72 hours post-infection (HPI). Apoplast fluid was sampled, and label-free proteomic analysis was performed via spectral scanning. The results reveal that barley leaf apoplastic fluid (48 HPI) becomes enriched with pathogenesis-related (PR) proteins such as chitinases, nsLTPs, thaumatin-like proteins, Bowman–Birk-type trypsin inhibitors, and leucine-rich repeat proteins, among other PR-related proteins. The development of an efficient apoplastic isolation method using barley allowed us to reveal these differences between F.g. infected plants and mock treatment. The proteins identified suggest potential resistance mechanisms specific to the apoplast which may impact the severity of FHB which develops in small grain cereals.