Tentoxin is an interesting non-specific toxin produced by some Alternaria species, known as pathogenic organisms. The high-performance liquid chromatographic determination of tentoxin by using a YMCA-312 column, a reversed type of octadecylsilica, with 0.05 M ammonium dihydrogenphosphate acetonitrile (7:3) as the mobile phase is described. The limit of detection is 0.1 pg/ml. One assay can be performed by using culture liquid without any other procedures within 30 min.