The development of a reliable in vitro feeding system has enabled the rapid testing of presumptive anti-mite factors, a mixture of protease inhibitors (PI), by incorporating these into the blood feed. Mites were collected from a caged-hen facility and underwent a regime of starvation under varying conditions of temperature and darkness to determine the optimum conditions that encouraged mites to feed in the in vitro system. The efficacy of two anti-coagulants, heparin (HS) and acid citrate glucose (ACD), on mite feeding rates and mortality was evaluated. The ability of a mixture of PI to kill mites was also evaluated. The rate of feeding was greater in mites that were starved and cooled for between 7 and 30 days compared with mites that were not starved or cooled. The percentage of mites that fed when presented with HS treated blood (70%) was greater when compared with ACD treated blood (48%). The mortality of mites fed blood treated with HS + PI increased to 45% compared with a mortality level of 5% in mites fed on blood treated with HS alone. A reliable in vitro method for feeding D. gallinae which has the potential to be used to rapidly screen blood-borne products for their efficacy in reducing mite numbers has been developed.