Reliable quantification of peptides and proteins is essential for drug discovery. We report the successful development and validation of an accurate and broadly applicable high performance liquid chromatography hyphenated to fluorescence detector procedure for the quantitative determination of the aromatic amino acids tyrosine, phenylalanine, and tryptophan, without relying on derivatization chemistry. Using ion-pair chromatography, fluorescent amino acids were clearly separated within 10 minutes. The hydrolysis of peptides was performed under acidic and heated conditions to yield the monomeric building blocks. Various protecting agents were tested to ensure tryptophan stability. The presented analytical method accurately (>95%) quantifies all fluorescent residues. The power of the method was confirmed by correct quantification of protein reference standard to 98.6% over all fluorescence traces. The method allowed us to identify pre-analytical differences between the nominal and actual concentrations of 12 peptide solutions. Salt formation, weighing errors, and other pre-analytical pitfalls resulted in noteworthy differences of up to 85% between the indicated and actual concentration of peptide solutions, subsequently leading to false positive or negative interpretation of activity data. Finally, only one solution is needed to perform quantification as well as UV-purity tests and can further be used as stock solution for activity testing.