Microwave-assisted extraction (MAE) followed by high performance liquid chromatography (HPLC) with a fluorescent detector (DAD) was used and developed for the quantitative analysis of the mycotoxin ochratoxin A (OTA) in commercial roasted coffee beans. This alternative approach has been compared with the conventional extraction that uses hydrogen carbonate aqueous solution followed by OchraTest immunoaffinity analysis. The effect of two experimental tunable MAE parameters (temperature and pressure) on the extraction efficiency of OTA have been investigated using coffee samples forti?ed at different contamination levels. The optimum extraction conditions were obtained using a temperature of 50 °C and a 500 W microwave power. OTA quantity extracted using MAE was similar to that obtained by conventional extraction from samples fortified at 5, 10, and 100 ng g^-1 levels. At a 20 ng g^-1 level, MAE was more effective than the conventional method. The MAE setting parameters have been optimized showing both extraction time and solvent consumption have been considerably reduced, retaining high OTA recovery values.