I have studied the separation of the mixture of three biogenic amines (tyramine, tryptamine and 2-phenylethylamine) using high-performance liquid chromatography without derivatization. These amines were used as a model compounds for biologically active compounds that are often used in the pharmaceutical industry. The main goal of this study was a comparison of two different separation methods: ion-pairing and mixed-mode chromatography. On each column, I observed the effects of buffer ionic strength, pH and polarity on separation. Selected biogenic amines were determined by spectrometric detector since they all absorb the light in the UV spectrum. Corresponding chromatographic methods for separation and determination of biogenic amines were developed and validated on the columns Primesep 200 and Kromosil 300-5-C18.