Methotrexate (MTX) is an antagonist of folic acid metabolism that was initially designed to treat malignancies, including childhood leukemia. After its anti-inflammatory properties were discovered, use of MTX became widespread in the treatment algorithms for several autoimmune illnesses, including the inflammatory bowel diseases (IBD), Crohn’s disease (CD) and ulcerative colitis (UC). The specific cause(s) of IBD in general, and CD in specific, have not yet been fully elucidated. However, most investigators agree that the pathogenesis is likely due to a combination of genetic vulnerability and precipitating environmental exposures. Some of these identifiable modifiers include adherence to a low fiber diet, vitamin D deficiency, smoking, and an alteration in the diversity of the gut microbiome in response to viral or bacterial illness or antibiotic medications. Disease activity in patients with CD, particularly during clinical trials, is assessed using composite indices, including the Crohn’s Disease Activity Index (CDAI), Crohn’s Disease Endoscopic Index of Severity (CDEIS), and the Short Inflammatory Bowel Disease Questionnaire (SIBDQ). A wide range of medications is used to induce and maintain long-term remission in patients with active CD. These include corticosteroids, immunosuppressive agents (thiopurines and MTX) and several classes of biologics such as TNF-inhibitors, anti-IL-12/23 and anti-adhesion molecules. TNF-inhibitors are often used as first-line biologic therapies in patients with IBD because they have been in widespread use for over two decades and therefore afford the clinician a more data-driven consideration of risk to benefit ratio when discussing treatment options with their patients. However, there is a relatively high rate of primary non-response and acquired secondary loss of response to TNF-inhibitors. A secondary loss of response often results from the production of neutralizing antibodies, referred to as Antibodies to Infliximab (ATI). In response, concomitant low-dose oral MTX therapy has been employed by clinicians to reduce the immunogenicity of biologic therapy. In addition, previous studies have also demonstrated the efficacy of MTX monotherapy in maintaining clinical remission in patients with CD when delivered at relatively higher doses. However, there is no consensus on the proper dosing or route of administration (oral or parenteral) of MTX. This knowledge gap has resulted in inconsistent clinical practice across physicians and institutions. Pharmacologic studies have identified the metabolic pathways underpinning the mechanism of action of MTX. It is generally understood that MTX in its native form is free to move across cell membranes in a bidirectional manner. It is only after MTX has been glutamated (MTX-PG) that it is “caged” within the cell and can exert its effects. MTX can be glutamated on up to five discrete sites, each contributing to its stereospecificity and membrane permeability. A better understanding of this process may inform the development of rational dosing and pharmacokinetic-based treatment algorithms that provide patients with a sufficient MTX (and subsequently MTX-PG) level required to achieve anticipated clinical efficacy but not so high as to contribute undo morbidity to treated patients. Therefore, optimizing MTX/MTX-PG dosing can significantly advance the utility of this immunomodulatory pathway to treat IBD and other autoimmune disorders. MTX can be administered orally or parenterally, the latter being delivered by either subcutaneous or intramuscular injection. Previous studies have demonstrated increased bioavailability of the drug at higher doses when delivered via the parenteral route. The native form of MTX has a short half-life and is eliminated from the body within 24 hours. Instead, it is the active metabolites of MTX which are retained for more extended periods of time and are ultimately responsible for the anti-inflammatory effects in the body. A MTX molecule can have anywhere from 1 and 5 glutamyl groups attached to it, denoted as MTX-PG1, MTX-PG 2, MTX-PG3, MTX-PG 4, and MTX-PG 5. The mechanism(s) by which MTX-PG moieties contribute anti-inflammatory activity is not fully understood but remains an area of active research. Several studies have explored the association between disease activity and erythrocyte MTX-PG levels. While initial results were mixed, more recent prospective cohort studies in patients with Rheumatoid Arthritis (RA) found an inverse relationship between intracellular levels of longer-chain MTX-PG (MTX-PG3, MTX-PG4, and MTX-PG5) and disease activity. This finding has raised the possibility that monitoring MTX-PG levels could be used as a clinical tool to optimize MTX therapy for patients. However, several key issues persist, including high interpatient variability in MTX-PG levels. Similar studies in patients with CD have thus far been scarce. More prospective studies are needed to explore the utility of MTX-PG pharmacokinetics as a useful biomarker and clinical tool to develop an individualized approach to managing patients with Crohn’s disease.