The potential of high pressure (HP) to affect the content of free amino acids (FAA) using seedlings of Brussels sprouts as a simple non-chopped vegetable system was examined. Firstly, the effect on FAA composition during growth was assessed and it was found that the composition of total free amino acids (TFAA) and individual FAA changed dramatically during growth of the seeds to the seedling at 7 days with the highest content of TFAA. Secondly, 7-day-old seedlings were HP-treated at various pressure levels (200–800 MPa for 3 min at 5 °C). As expected the HP-treatment did not affect the amino acids as no changes in TFFA were found immediately after pressurisation. In this line, HP-treatment up to 800 MPa had minor, but significant, effect on the FAA concentrations of 10 FAA (Ala, Asp, Glu, Gly, Leu, Phe, Pro, Ser, Trp and Tyr) and no significant changes were found for 7 of the FAA (Asn, Gln, His, Ile, Lys, Thr, and Val) concluding that the short pressure time (3 min) was insufficient to activate indigenous proteolytic enzymes. Furthermore, changes in the FAA content and composition of HP-treated seedlings during storage (0, 1, 2, and 4 days at 4 ± 2 °C) were evaluated in order to assess changes in the proteolytic enzyme activity. It was found that the changes in FAA differed according to the specific amino acids as well as the HP processing conditions and the subsequent storage time. These results suggest that HP treatment affects proteolysis and/or certain amino acids metabolism pathways in Brussels sprouts seedlings after HP treatment and during subsequent storage.