Asthma often begins in childhood, although making an early diagnosis is difficult. Clinical manifestations, the exclusion of other causes of bronchial obstruction, and responsiveness to anti-inflammatory therapy are the main tool of diagnosis. However, novel, precise, and functional biochemical markers are needed in the differentiation of asthma phenotypes, endotypes, and creating personalized therapy. The aim of the study was to search for metabolomic-based asthma biomarkers among free amino acids (AAs). A wide panel of serum-free AAs in asthmatic children, covering both proteinogenic and non-proteinogenic AAs, were analyzed. The examination included two groups of individuals between 3 and 18 years old: asthmatic children and the control group consisted of children with neither asthma nor allergies. High-performance liquid chromatography combined with tandem mass spectrometry (LC-MS/MS technique) was used for AA measurements. The data were analyzed by applying uni- and multivariate statistical tests. The obtained results indicate the decreased serum concentration of taurine, L-valine, DL-β-aminoisobutyric acid, and increased levels of ƴ-amino-n-butyric acid and L-arginine in asthmatic children when compared to controls. The altered concentration of these AAs can testify to their role in the pathogenesis of childhood asthma. The authors’ results should contribute to the future introduction of new diagnostic markers into clinical practice.