The consumption of bee pollen has increased in the last few years due to its nutritional and health-promoting properties, which are directly related to its bioactive constituents, such as amino acids. Currently, there is great interest in understanding the role of these in bee products as it provides relevant information, e.g., regarding nutritional value or geographical and botanical origins. In the present study, two fast chromatographic methods were adapted based on commercial EZ:faast™ kits for gas chromatography-mass spectrometry and liquid chromatography–mass spectrometry for determining free amino acids in bee pollen. Both methods involved the extraction of amino acids with water, followed by a solid phase extraction to eliminate interfering compounds, and a derivatization of the amino acids prior to their chromatographic separation. The best results in terms of run time (<7 min), matrix effect, and limits of quantification (3–75 mg/kg) were obtained when gas chromatography–mass spectrometry was employed. This latter methodology was applied to analyze several bee pollen samples obtained from local markets and experimental apiaries. The findings obtained from a statistical examination based on principal component analysis showed that bee pollen samples from commercial or experimental apiaries were different in their amino acid composition.