Green coffee and its derivatives, which include beverages, dietary supplements, additional foods, and nutraceuticals, are appreciated for their potent antioxidant properties. However, there is a significant risk of the contamination of green coffee and its products by fungi and their mycotoxins, particularly ochratoxin A (OTA). This contamination can happen in traditional and organic green coffee during various stages, such as berry picking, crop storage, and transportation. Ochratoxin A (OTA) is known to have harmful effects on the kidneys (nephrotoxicity), liver (hepatotoxicity), and nervous system (neurotoxicity) and can cause congenital disabilities (teratogenicity) and cancer. This work validated a chromatographic method to measure OTA levels in green coffee following the UNI CEI EN ISO/IEC 17025: 2018. The analytical method's validation is essential to guarantee data traceability and avoid measurement errors. The experimental design involved four steps: extraction with polar solvents, elimination of interfering substances using an affinity column, toxin separation by reversed-phase chromatography, and spectrophotometric identification of toxins. The validation process demonstrated that the method can recover OTA levels (76%) while conforming with the performance criteria required for toxins by Regulation (EU) no. 519/2014. The regression coefficient (r-value = 1) and residual analyses proved the linearity of the calibration curve; LOD, LOQ, and the measuring range confirmed that the test ​​detects the minimum OTA levels permitted by the current legislation in coffee (3 µg/kg - 5 µg/kg). The test's precision (with a standard deviation of 0.0073) and accuracy (within ± 0.64 µg/kg) were considered statistically satisfactory. The method's results were trustworthy, with a confidence level of 95%, based on uncertainties determined through the metrological approach. Therefore, the analytical method was considered sensitive, precise, accurate, and suitable for determining ochratoxin concentrations in compliance with the regulations in force.