Abstract The AccQ.Tag™ method, as a well-established protocol for amino acid analysis combining derivatization procedure, dedicated HPLC separation and fluorescence detection, was properly transferred and accordingly optimized for the application on ultra performance liquid chromatography (UPLC™) and UV detection. Capitalizing on sub-2 μm particles, this newly established UV-UPLC™ technique facilitated efficient chromatographic separation of 21 amino acid derivatives within 12 min. In addition, UPLC™ demonstrated significant improvements due to superior performance and reduced run times compared with the former 35 min of the original HPLC protocol. Using UV instead of fluorescence detection, amino acid quantification after pre-column derivatization with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) yielded appropriate sensitivities within the low pmol range with corresponding detection limits varying from 0.11 to 0.57 pmol per injection. Moreover, the UPLC™ method was applied to characterize changes in the free ( FAA) as well as total amino acid ( TAA) profiles specific to culture media at three distinctive stages of fermentation: fresh medium, fermentation broth after cell mass production prior to induction and after product expression at the end of fermentation. Amino acid profiles intrinsic to the fresh, sterilized medium indicated free, hence more bioavailable, amino acids at a FAA/ TAA ratio of 40%, whereas ongoing fermentation implied a rather specific, successive decline in selective FAA concentrations. Thereby, the most distinctive variations in FAAs were highlighted by aspartic acid, serine and threonine, each exhibiting an almost complete uptake from the culture media (−96% to −99%), with remaining FAA/ TAA ratios of 1%, 8%, and 1%, respectively. This indeed may indicate limitations and shortages within the nutrient broth. Thus, amino acid monitoring utilizing high-throughput chromatography, such as UPLC™, can be considered as a valuable tool to facilitate rapid adjustments of fermentation broths and to optimize culture media to specific requirements.