A method was developed to quantitate the glucuronide conjugates (phase II metabolites, log P = 0.6-1.8) of the phase I hydroxylated metabolites (log P = 2.6-2.7) of beta-arteether (2) (log P = approximately 4.0) in rat plasma and urine. The biological fluids were extracted with a C-18 solid-phase cartridge, then subjected to analysis by thermospray high-performance liquid chromatography/mass spectroscopy with a C-18 reversed-phase analytical column in the selected ion monitoring mode. The detection limits for these glucuronide conjugates in plasma and urine ranged from 25 to 60 ng/mL, and the detection limits were appropriate for the biological activity of these glucuronide conjugates in plasma. Following intravenous administration of arteether (11.6 mg/kg), the formation of these glucuronide conjugates in rat urine was undetected (below detection limit) over 24 h. However, in rat plasma, formation of 9alpha-hydroxyarteether glucuronide (13; 255 ng/mL, IC50 = 6560 ng/mL, log P = 0.93) was noted at 15 min post-injection. Although several of the glucuronide metabolites of arteether retained sufficient in vitro antimalarial activity to be potentially useful antimalarial agents if administered as prodrugs, the levels of the glucuronides formed from arteether were too low to contribute to the antimalarial activity observed for the parent compound.