A rapid, sensitive, and reproducible pre-column derivatisation procedure has been established for the simultaneous determination of 20 amino acids by high-performance liquid chromatography using fluorescence detection. The amino acids were derivatized using o-phthalaldehyde and 9-fluorenylmethyl-chloroformate reagents. The optimal conditions for simultaneous separation and detection of both primary and secondary amino acids were investigated. The developed method has several advantages, namely automated pre-column derivatization, short analysis time with optimal separation, a simple and economical mobile phase, high level of precision for peak area and retention time, and higher sensitivity with more reliability of peak identification. The biological media development is the key parameter for macromolecule drug discovery. Biological media amino acids in three consecutive discovery batches were determined and the results showed a good agreement with hypothetical value. The method appears suitable for application to measure biological media amino acids at various stages of macromolecule drug discovery.