A Klun & Debboun (K&D) test module, previously developed and used for quantitative measurement of the efficacy of mosquito repellents on human volunteers, was adapted for in vitro evaluation of repellents by coupling the module with a membrane-blood reservoir. Performance of Deet, Bayrepel^®, and SS-220 insect repellents in the new in vitro system was compared with their performance on humans against mosquitoes using our standard in vivo system. For each compound, in vitro dose–response assays were conducted with compounds applied to cloth positioned over blood reservoirs covered with Baudruche membrane against Aedes aegypti. The repellents were also tested in vitro against Anopheles stephensi and Ae. aegypti at a fixed dose of 24 nmol compound/cm² cloth over the Baudruche and Edicol collagen membranes. Concurrently, the repellents were tested at the fixed dose using the K&D module on human volunteers. The observed proportions of both mosquito species deterred from biting in the fixed doses in the in vitro assays were similar to those obtained using humans, being clearly able to distinguish controls from repellents, and differing only in the ranking of the effectiveness of some of the repellents. Dose–response relationships of the in vitro and in vivo systems were also very similar, although not directly comparable because the data were not collected concurrently. This new in vitro assay system can be used in high throughput screening of compounds to identify new repellents having potential for use as topical mosquito repellents on humans.