Mycotoxins are toxic secondary metabolites of fungi. They can contaminate food and cause harmful effects both in animals and humans and therefore cause serious problems for human economy and health. In the organism they usually enter through ingestion of contaminated food, but can also enter through the respiratory tract. Ochratoxin A is one of the most important mycotoxins, a product of fungi from Aspergillus and Penicillium. It is classified as a category 2B carcinogen and has primary adverse effect on the kidneys. High performance liquid chromatography associated with mass spectrometry due to its high sensitivity and selectivity proved to be an excellent method of analysis. A method using the mass spectrometer with ion trap analyzer and electrospray ionization technique has been shown as particularly suitable for MSn spectrum analysis. The goal of this study was to optimize the conditions for identification and analysis of ochratoxin A, as well as to describe its fragmentation as accurately as possible. First, the optimum drying gas flow rate was selected for the ionization of the sample, followed by fragmentation and MSn analysis using a mass detector. Characteristic fragments were identified and a fragmentation path was described, based on which identification of an analyte or identity of a compound in a sample could be performed. Two main pathways of OTA fragmentation are assumed. In the first pathway, the cleavage of the amide bond and formation of the m/z 239 fragment occurs. In the second proposed fragmentation pathway loss of water (m/z 386) occurs first, followed by loss of the carbonyl group resulting in formation of the m/z 358 fragment. Subsequently, ammonia is removed and lost, and from the resulting m/z 341 fragment, the characteristic m/z 257 fragment is formed. The loss of water again yields to the m/z 239 fragment which is also characteristic for the first proposed fragmentation pathway. Further analysis showed that the m/z 239 fragment gradually cleaved to the m/z 193 fragment.