The infectious dose (ID) of an inoculum for which 50% of susceptible mice will become infected (ID(50)) with Orientia tsutsugamushi is usually determined by rechallenging mice that have already been challenged with O. tsutsugamushi to determine the lethal dose (LD)(50) titer of the inoculum. Those mice not killed by the initial challenge and which survived a rechallenge with 1000 LD(50) were considered immune and to have been initially infected with O. tsutsugamushi. Mice that succumbed to the rechallenge were considered not to have been initially infected. We have developed a more rapid method of determining the ID(50) of inocula for use in our vaccine studies based upon the observation that mice surviving initial challenge and that go on to survive rechallenge produced detectable IgG to O. tsutsugamushi antigens by enzyme-linked immunosorbent assay (ELISA). Mice that did not survive rechallenge, and therefore did not receive an initial infectious inoculum did not produce detectable IgG to O. tsutsugamushi antigens. Both original LD(50) and ID(50) titers determinations require observation of mice for 21 days post-challenge. Our new ID(50) determination does not require mice or the additional 21-day observation period for rechallenge and therefore is more rapid and cost-effective than the previous standard method of determining ID(50) titer necessary for the evaluation of vaccine candidates.