In this research, we developed and validated a high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method to determine Ochratoxin A (OTA) levels in coffee powder samples. Separation of Ochratoxin A was achieved using a Hypersil XDB-C18 column (2.1 mm x 100 mm) with a particle size of 1.8 µm, coupled with tandem mass spectrometry. The mobile phase, composed of acetonitrile: water: acetic acid in a ratio of 80:20:1 (v/v/v), operated at a flow rate of 0.1 mL/min. Each sample underwent a total chromatographic analysis time of approximately 5 minutes, with Ochratoxin A eluting at a retention time of 3.259 minutes. The linear range spanned from 1 to 500 ng/mL, characterized by a linear regression equation (Area = C + 19521) with a high correlation coefficient (r = 0.9991). The limit of detection (LoD) and limit of quantification (LoQ) for Ochratoxin A were established at 0.63 µg/mL and 2.11 µg/mL, respectively. Recovery rates for Ochratoxin A ranged from 84.86% to 94.54%. The Ochra Test WB solid phase extraction column was chosen to purify the extract OTA from a coffee sample. Among the 55 coffee powder samples analyzed, 9 samples (16.36%) were found to be contaminated with OTA. Notably, all Ochratoxin A-contaminated samples fell within the permissible limits set by the Ministry of Health Vietnam, international organizations. While the concentrations of OTA in the studied samples did not surpass the maximum residue limit, but the presence of a positive result indicates pollution of coffee by these toxic substances.