Therapeutic drug monitoring (TDM) can improve clinical care when using drugs with pharmacokinetic variability and a narrow therapeutic window. Rapid, reliable, and easy-to-use detection methods are required in order to decrease the time of analysis and can also enable TDM in resource-limited settings or even at bedside. Monitoring methotrexate (MTX), an anticancer drug, is critical since it is needed to follow the drug clearance rate and decide how to administer the rescue drug, leucovorin (LV), in order to avoid toxicity and even death. We show that with the optimized nanopillar-assisted separation (NPAS) method using surface-enhanced Raman scattering, we were able to measure MTX in PBS and serum in the linear range of 5-150 μM and confirmed that MTX detection can be carried out even in the presence of LV. Additionally, when NPAS was combined with centrifugal filtration, a quantification limit of 2.1 μM for MTX in human serum sample was achieved. The developed detection method enables fast detection (10 min) and quantification of MTX from human serum (>90% accuracy). Furthermore, we show the potential of the developed method for TDM, when quantifying MTX from clinical samples, collected from patients who are undergoing high-dose MTX therapy.