Acute tryptophan depletion (ATD) is a method of reducing central nervous serotonin (5-HT) synthesis in humans by administering an amino acid (AA) beverage lacking in tryptophan (TRP), the physiological precursor of 5-HT. However, to date, the use of conventional ATD protocols in children and adolescents was limited due to frequently observed side effects (e.g., vomiting and nausea). This study investigated the effects of diminished central nervous system 5-HT synthesis on plasma concentrations of relevant AAs and TRP influx into the brain in 24 healthy young adults using the ATD procedure Moja-De, a test protocol that has been used in preliminary research in youths. Twenty-four healthy participants received ATD and a TRP-balanced amino acid load (BAL) using a randomized double-blind within-subject crossover design. Plasma concentrations of the relevant AAs that compete with TRP on the same transport system were assessed at baseline and 90, 180, and 240 min after ATD/BAL intake. TRP influx across the blood-brain barrier was calculated using Michaelis-Menten kinetics with a correction for multiple substrate competition, indicating a significant decrease in TRP influx into the central nervous system under Moja-De. ATD Moja-De decreased TRP influx into the brain and central nervous system 5-HT synthesis safely and effectively and was well tolerated, allowing it to be used in children and adolescents. Future research into other secondary, compensatory effects induced by ATD in patients with neuropsychiatric disorders and healthy populations is needed. ATD Moja-De allows this type of research with a focus on a developmental viewpoint.