Abstract: This paper describes a bioassay method for the determination of ansamitocin titers. A fungal strain sensitive to ansamitocin was classified to the genus Trichoderma based on phylogenetic analysis of its ITS sequence, and designated as Trichoderma CPCC 400749. PDA plates of Trichoderma CPCC 400749 were prepared to assay ansamitocin titers of Actinosynnema pretiosum ATCC 31565. The titers were consistent with those determined by HPLC. The bioassay method may have the potential use in high-throughput screening for Actinosynnema pretiosum mutants with improved ansamitocin titers.