The content of free and hydrolyzed primary amino acids was determined for five species of microalgae: Haematococcus pluvialis, Skeletonema costatum, Arthrospira sp., Acutodesmus acuminatus, and Botryococcus braunii. The amino acid profile was determined using an automated HPLC-DAD reverse phase pre-column analysis method, which is described in detail. Acid hydrolysis using 6 M HCl and autoclaving at 120 °C for 2 h was validated using casein of bovine origin as standard. Nitrogen to protein conversion factors were calculated for each microalgae. Haematococcus pluvialis contains the least amount of free amino acids (38.8 ± 0.15 mg AA/100 g DW), while Arthrospira sp. shows the most enriched free amino acid profile (316.1 ± 0,11 mg AA/100 g DW). Skeletonema costatum shows the least amount of amino acids after hydrolysis with 5.09 ± 0.28 g AA/100 g DW while Arthrospira sp. achieves 27.76 ± 0.19 g AA/100 g DW. The total protein content and its relationship with the detected amino acid content are discussed.