Sixty times of high dose methotrexate (MTX) with citrovorum factor rescue (HDMTX-CF) therapy were applied for 15 patients with osteosarcoma. The serum level of MTX was measured at every 24 hr. by FPIA (Fluorescence Polarization Immuno Assay) method and blood analysis was performed at the same time. Fourtysix parameters were measured in each HDMTX-CF therapy and examined by multiple parameter statistical analyses by a personal computer. In the results, the number of performed chemotherapy, rescue time, serum GOT and creatinine level at 24 hr. were the most relative factors for the change of serum MTX level.