Background: High-dose methotrexate therapy is an effective treatment for primary central nervous system (CNS) lymphoma and other tumors. Methotrexate (MTX) is an antimetabolite which penetrates blood brain barrier when administered intravenously at high doses. Defective or delayed clearance of MTX may lead to toxicities, especially acute kidney injury (AKI) in 2-13% of patients. We developed a protocol to mitigate side effects by checking MTX levels every 24 hours, administering leucovorin or glucarpidase rescue, and hydration with urine alkalization. This study examines the risk factors for developing AKI after high dose MTX administration. Methods: Patients who began treatment with high dose methotrexate in 2020 and 2021 were eligible. We collected data on demographics, diagnosis, methotrexate levels, laboratory levels at baseline and after treatment, toxicities, interventions and outcomes. Results: 26 patients were included, who received 112 cycles of treatments (median 3.5, range 1-10). Median age was 62 years (range 25-83) and 57% were male. 20 patients experienced elevated MTX levels. Among the 112 cycles, MTX levels were elevated at 24h, 48h and 72h in 22 (19.6%), 16 (14.3%), and 45 (40.2%) of the cycles respectively, which trigged an increase in the rescue leucovorin dose and hydration dose in 26 (23.2%) and 16 (14.3%) cycles, respectively. AKI was recorded in 20 cycles (17.9%), with 17 (85%) being grade 1 (ULN – 1.5 x ULN), and 3 (2.7%) being grade 2 (1.5-3x ULN). In 20 cycles with AKI, 9 (45%) had grade 1 elevated creatinine level pretreatment; 8 (88.9%) of them remained grade1 without increase > 0.2. The development of AKI occurred in all MTX dose levels, without a difference in frequency. 57% of cycles with elevated 24h MTX level, and 13% of cycles with delayed clearance > 4 days, were associated with AKI. 75% of cycles with AKI had delayed clearance (avg 7 days). Two patients required glucarpidase rescue treatment, based on elevated 24-36 h MTX level and grade 1-2 AKI; one of these patients permanently discontinued MTX. Conclusions: Among 26 patients who received a total of 112 cycles of high dose MTX treatment, 11 developed AKI in 20 cycles, mostly grade 1 with spontaneous recovery. MTX level elevation at 24 hours predicted the development of AKI. Elevated baseline creatinine or MTX dose range is not a risk factor for AKI.