Background <p>High-dose methotrexate (HDMTX) is a key treatment for lymphoma with central nervous system involvement. Whether incorporating cystatin C into glomerular filtration rate estimation improves methotrexate (MTX) clearance prediction remains unclear.</p> Objectives <p>We aimed to evaluate whether cystatin C-inclusive glomerular filtration rate equations improve MTX clearance prediction and to explore the relationship between MTX exposure and acute kidney injury (AKI) in adult patients with lymphoma receiving HDMTX.</p> Methods <p>This was a prospective single-center study performed on 80 adult patients with lymphoma receiving HDMTX (1.5–8 g/m²) over a 4-h infusion. A population pharmacokinetic model was constructed using data from 80 administrations of HDMTX and 427 serum MTX concentrations. The population pharmacokinetic model estimated MTX concentrations were included in a logistic regression to assess the relationship between MTX exposure and AKI.</p> Results <p>A two-compartment model best described the pharmacokinetic data, with baseline albumin and CKD-EPI creatinine-cystatin C (eGFRCr-CysC) as significant covariates on clearance. Seventeen patients (21%) developed any-stage AKI. Among those receiving ≤&#xa0;3.5 g/m², model-estimated 4-h MTX concentrations were associated with AKI (odds ratio: 1.02 per µmol/L; <i>p</i> = 0.0038), with an optimal threshold of 160 µmol/L (area under the concentration–time curve: 0.818). Patients above this threshold were 22 times more likely to experience AKI (<i>p </i>= 0.0005). This association was not observed in patients treated with 8 g/m². Despite a lower dose and exposure, patients receiving ≤&#xa0;3.5 g/m² demonstrated a stronger concentration–toxicity relationship.</p> Conclusions <p>Our results support the use of cystatin C-inclusive glomerular filtration rate estimates in MTX pharmacokinetic modeling and suggest early MTX concentration sampling may identify AKI risk, enabling proactive, AKI-mitigating clinical interventions during HDMTX therapy.</p>