Background and objectives: Methotrexate (MTX) is a chemotherapeutic agent that functions as a folic acid antagonist. The frequency of high dose methotrexate (HDMTX)-associated toxicity is variable. In this study, we investigated the frequency of myelotoxicity and hepatotoxicity 7 days after HDMTX infusion. Patients and methods: This study included children diagnosed with acute lymphoblastic leukemia (ALL) between January 2010 and April 2015. The patient blood counts and biochemical parameters measured before and after 7 days of HDMTX infusion were retrospectively recorded. We assessed HDMTX infusions for 48 children. The number of patients and drug doses included the following: 17 children receiving 1 g/m2 (68 infusions), 14 children receiving 2 g/m2 (56 infusions), and 17 children receiving 5 g/m2 (68 infusions). The classification of toxicity was made based on the Common Terminology Criteria for Adverse Events (CTCAE) 2010 criteria. Myelotoxicity was defined as a hemoglobin level .05, for all). There was no correlation between hematologic parameters and transaminase levels and MTX levels at 42 h. Conclusion: Hematologic toxicity was the most common toxicity observed. The data indicate the hematologic toxicity increased after repeated cycles in patients receiving 5 g/m2. However, the hepatic toxicity decreased with additional cycles. Our results show the level of MTX at 42 h is not effective to identify toxicity.