Antimalarial drugs such as chloroquine are believed to act by inhibiting hemozoin formation in the food vacuole of the malaria parasite. We have developed a new assay for measuring and detecting inhibition of synthetic hemozoin (beta-hematin) formation. Aqueous pyridine (5% v/v, pH 7.5) forms a low-spin complex with hematin but not with beta-hematin. Its absorbance obeys Beer's law, making it useful for quantitating hematin concentration in hematin/beta-hematin mixtures, allowing compounds to be investigated for inhibition of beta-hematin formation. The assay is rapid (60 min incubation) and requires no centrifugation. The beta-hematin inhibition data show good agreement with alternative assay methods reported by four laboratories. The assay was adapted for high-throughput colorimetric screening, allowing visual identification of beta-hematin inhibitors. In this mode, the assay successfully detected all 18 beta-hematin inhibitors in a set of 47 compounds tested, with no false positive results. The quantitative in vitro antimalarial activities of a set of 13 aminoquinolines and quinoline methanols were found to correlate significantly with beta-hematin inhibition values determined using the assay.