Chemical genetics is an emerging field that takes advantage of combinatorial chemical and small molecule libraries to dissect complex biological processes. Here we establish a fluorescence-based assay to screen for inhibitors of iron uptake by mammalian cells. Using this approach, we screened the National Cancer Institute's Diversity Set library for inhibitors of non-transferrin bound iron uptake. This screen identified 10 novel small molecule inhibitors of iron transport with IC(50) values that ranged from 5 to 30 microM. Of these ten compounds, only two blocked uptake of iron mediated by transferrin. Thus, this study characterizes the first small molecule inhibitors that distinguish between different pathways of iron transport.