The profile of amino acids and mono- and disaccharides in conventional polyfloral honey originated from Latvia and Tajikistan and less found in nature bumblebee honey from Russia was investigated. The analysis of free amino acids (FAAs) accomplished by multiple reaction monitoring (MRM) using triple quadrupole mass selective detection (HPLC-ESI-TQ-MS/MS) revealed the presence of 17 FAAs. The concentration of FAAs varied in the range of 0.02–44.41 mg 100 g⁻¹ FW. Proline was the main representative of FAAs, contributing to the total amount of FAAs from 41.7% to 80.52%. The highest concentration of proline was found in bumblebee and buckwheat honey, corresponding to 44.41 and 41.02 mg 100 g⁻¹, respectively. The concentration of essential amino acids (AAs), i.e., leucine, and isoleucine was found to be the highest in buckwheat honey contributing up to 12.5% to the total amount of FAAs. While, the concentration of branched-chain AAs fluctuated within the range of 1.08–31.13 mg 100 g⁻¹ FW, with buckwheat honey having the highest content and polyfloral honey the lowest, respectively. The results of this study confirmed the abundance of FAAs both in honeybee and bumblebee honey. However, the concentration of individual FAAs, such as proline, aspartic acid, leucine, and isoleucine in bumblebee honey was many folds higher than observed in honeybee polyfloral honey.