In this prospective intention-to-diagnose pilot study, we aimed to assess accuracy of serum and fecal amino-acids to discriminate de novo pediatric inflammatory bowel disease (IBD) and non-IBD children. Patients with suspected IBD were allocated the IBD (n = 11) or non-IBD group (n = 8) following laboratory testing or endoscopy according to the revised Porto-criteria. Fecal calprotectin levels were obtained, an additional blood and fecal sample were collected. Fecal and serum amino-acid profiles were analyzed using high performance-liquid chromatography. Nine fecal amino-acids (alanine [area under the curve 0.94], citrulline [0.94], glutamine [0.89], leucine [0.98], lysine [0.89], phenylalanine [0.99], serine [0.91], tyrosine [0.96], and valine [0.95]) differed significantly between IBD and non-IBD. In serum, no significant differences were observed. This study underlines the potential of fecal amino-acids as novel, adjuvant noninvasive, and low-cost biomarkers in the diagnostic work-up of pediatric IBD detection.