The objective of the study was to obtain a natural α- and β-carotene preparation from carrot. The squeezed juice was coagulated with a concentrated hydrochloric acid at about 95 °C. The sedimentation was carried out at a temperature close to 0 °C, and the coagulum was centrifuged. Next, α- and β-carotene were extracted from this centrifuged coagulum using an extraction mixture: petroleum ether & acetone (1:1 v/v). The extract produced was vacuum-filtered and chemically dehydrated. The extraction mixture was evaporated under vacuum, and the crystallization of carotene was carried out at -20°C. The crystals of α- and β-carotene were isolated on a filter paper by washing them with hexane; then, they were dried under vacuum. From the amount of 50 kg of carrot, more than 2 g of the crystal-formed α- and β-carotene preparation were obtained on average. The HPLC chromatographic analysis proved that the preparation produced was characterized by high purity and by α- to β-carotene ratio being 1:1.7. Additionally, the identification of α- and β-carotene was confirmed on the basis of UV/VIS spectra (diode array detector) and mass spectrum obtained using a liquid chromatography coupled with mass spectrometry, and with the ionization using electrospray (LC-ESI/MS).