Background and study aim: Methotrexate (MTX) as an antineoplastic agent inhibits dihydrofolate reductase. The frequency of high dose methotrexate (HDMTX) associated toxicity is variable. In this study we investigate the frequency of myelosuppression following 5 and 9 days of HDMTX infusion and MTX delayed elimination in subsequent MTX cycles in children with Acute lymphoblastic Leukemia (ALL). Patients and Methods: This study included 28 children diagnosed with ALL during the period between May2014 to April 2016. Complete blood counts were measured before and after 5 and 9 days of HDMTX infusion and MTX levels at 42h in 28 children with ALL. The HD-MTX dose is 5 gm/m2 during 102 infusion of HD MTX at consolidation phase of ALL therapy. The MTX levels at 42 h in patients with and without toxicity were compared to evaluate the correlation between MTX levels and myelotoxicity. Results: MTX infusion induced significant reduction in levels of TLC, ANC, RBCs, Hb and significant elevation of PLT count after 5 days of MTX administration. Additionally, after9 days of MTX infusion, there is significant decrease in TLC, ANC, and RBCs levels. However, no significant difference in the PLT count and Hb level occurred. There is gradual decrease in myelotoxicity after 5 days and increase after 9 days of MTX administration with regard to MTX cycles. There is no statistical difference in MTX level at 42 h between patients with and without myelotoxicity after 5 and 9 days of MTX infusion. MTX delayed elimination observed in MTX cycles 1, 2, 3 and 4 was 42.8% (n=12), 42.8% (n=12), 57.1% (n=16) and 72% (n=13) respectively. Conclusion: Myelotoxicity was decreased after 5 days of MTX administration and increased after 9 days with regard to MTX cycles. There is no correlation between MTX plasma concentration after 42 h and hematologic toxicity. Therefore, we cannot depend on MTX levels at 42 h to anticipate and predict hematologic toxicity.