Experimental trials in the natural host are essential for development and screening of effective vaccines. For chronic diseases of livestock such as paratuberculosis, these can be lengthy and costly in nature. An alternative is to screen vaccines in vitro; however, previous studies have found that vaccine success in vitro in existing screening assays does not translate to in vivo efficacy. To overcome these issues, we have developed a system that combines both in vivo and in vitro aspects. We hypothesise that the effectiveness of vaccine-induced immune responses mounted in vivo could be gauged by assessing the ability of immune cells to ‘control’ an in vitro infection. Monocytes from Merino wethers (n = 45) were infected with Mycobacterium avium subspecies paratuberculosis (MAP) in vitro, cultured with autologous lymphocytes and remaining viable intracellular MAP was quantified. Cells from MAP exposed sheep had a higher capacity to kill intracellular MAP compared to non-exposed controls (P = 0.002). Importantly, cells from MAP exposed uninfected sheep had a greater capacity to kill intracellular MAP compared to vaccinated animals that were infected (ineffective vaccination), indicating that this in vitro assay has the potential to gauge actual protectiveness, or lack thereof, of a vaccine.