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Aim: To determine the concentration of 6-thioguanine nucleotide (6-TGN) and 6-methylmercaptopurine (6-MMP), the interpatient variability, and the relationship with disease activity in patients with Chron’s disease on long-term stable doses of azathioprine (AZA). Methods: This is a prospective, tertiary care single-center hospital study in adult Chron’s disease patients treated with AZA. The quantification of phenotypic thiopurine methyltransferase enzyme activity in red blood cells and the estimation of the concentration of 6-TGN and 6-MMP in whole blood was performed using the HPLC-UV detector method. A clinical response was categorized as remission (Harvey-Bradshaw Index [HBI] < 5) or improvement (drop from baseline of at least three points of HBI) based on HBI. Exposure to metabolite concentrations and the clinical response to AZA treatment was observed. Results: Study analysis included 30 patients who were initiated on AZA, and they were followed up with an estimation of metabolite concentrations to determine their clinical outcome. At six months, 93% of ( n = 28) patients continued to be on AZA and had clinical improvement. All the patients achieved remission of Chron’s disease. Only two patients developed adverse effects such as joint pain and thrombocytopenia. Conclusion: AZA is a safe and effective therapy in managing Chron’s disease when administered after determining thiopurine methyltransferase phenotype and with dose optimization performed using therapeutic drug monitoring of 6-TGN and 6-MMP.