Methotrexate-d3 is primarily used as an internal standard in analytical chemistry for the quantification of methotrexate in biological samples. It is especially valuable in pharmacokinetic studies and therapeutic drug monitoring, where precise measurement of methotrexate levels is critical for optimizing dosage and minimizing toxicity. The deuterated form ensures accurate and reliable results by reducing interference from endogenous compounds during mass spectrometry analysis. Additionally, it aids in research focused on understanding the metabolism and distribution of methotrexate in the body, contributing to improved treatment strategies for conditions such as cancer, rheumatoid arthritis, and psoriasis.