The present paper reports the application of an optimized pre-column derivatization procedure with aminoquinolyl-N-hydrosysuccinimidyl carbamate (AQC) to the determination of amino acids levels in musts and wines using a quaternary HPLC eluent system, a Luna C 18 bonded silica column (250 × 4.6 mm) with a particle size of 5 μm and working temperature of 34 °C. The method permits the optimum separation of 21 amino acids and ammonium in musts and wines, with a relative standard deviation ranging from 2 to 4.5% in peak areas and lower than 1% for the retention time. Accuracy was lower than 5% for all the amino acids except glutamic acid, glutamine, γ-amino butyric acid and phenylalanine, which had an accuracy that varied from 5 to 10%. Excellent response linearity was obtained in the concentration range between 50 fmol and 14 μmol for all amino acids except cysteine which was in the 600 fmol to 14 μmol range. The method reported can be applied to the determination of amino acids in white musts and wines samples with no matrix effect. It was applied to the determination of 20 amino acids and ammonium in must and wine of the Airen variety Origin Denomination La Mancha (Spain). Arginine and proline are the majority amino acids in the must with concentrations of 214.6 and 122.05 mg L−1, respectively. Methionine and glycine have the lowest concentrations (1.52 and 2.54 mg L−1). Proline and arginine still have the highest concentrations in the wine (94 and 33.77 mg L−1) and isoleucine and leucine the lowest with 0.46 and 0.90 mg L−1, respectively.