Honeydew honey is produced by the honey bees from sweet sap (honeydew) secreted by plants or different types of insects that live on latifoliae and coniferous trees. Specific types of honeydew can be produced without the participation of insects. Bearing in mind that the chemical composition of honey primarily depends on the origin of honeydew/nectar, the aim of this study was to determine the amino acid composition of honeydew in order to determine possible specificities in the proportions of individual amino acids depending on the origin of the honeydew. For the purposes of determination of the botanical origin, a melissopalynological and sensory analysis was carried out, and selected physico-chemical quality parameters were determined. Out of 13 analysed samples, 7 honeydews originate from honeydew from coniferous trees or it predominates in the samples, and 6 samples originate from honeydew from latifoliae trees. The HPLC method with prior derivatization with o-phthaldialdehyde (OPA) and 9-fluorenylmethyl chloroformate (FMOC-Cl) was used for the determination of amino acids. The most abundant amino acids in honeydew are proline, phenylalanine, aspartic acid, glutamic acid and cysteine. The least represented are histidine, lysine, methionine, tryptophan and leucine. A higher total proportion of amino acids is observed in honeydews from latifoliae honeydew compared to honeydews from coniferous honeydew. In terms of total amino acids, the Quercus frainetto honeydew honey stands out with 734.70 mg/kg, while the total amino acids content in the other tested samples was 114.57 - 378.08 mg/kg.