Background Pediatric tuberculosis (TB) diagnosis is often challenging due to difficultly obtaining diagnostic respiratory specimens, which may contain low concentrations of Mycobacterium tuberculosis (Mtb) bacilli. However, we have reported that an immuno-affinity liquid chromatography-tandem mass spectrometry TB assay (ILM-TB) that detects a peptide derived from Mtb 10-kDa culture filtrate protein (CFP10pep) can diagnose TB regardless of patient age or infection site when analyzing trypsin-digested blood samples. Methods Discovery and validation cohorts were respectively generated from children who consecutively presented at two hospitals in South Africa (SA) from April 2012 to August 2017 (157 children <13 years-of-age) or at two communities in the Dominican Republic (DR) from July 2019 to May 2023 (101 children <17 years-of-age). All children were evaluated for TB at enrollment and 6-months post-enrollment, and assigned confirmed, unconfirmed, or unlikely TB diagnoses based on the 2015 NIH criteria for pediatric TB. Serum samples were collected at and two and six months after SA/DR enrollment, and at two weeks DR post-enrollment (270 SA and 304 DR samples) to evaluate ILM-TB assay performance. Findings SA and DR serum ILM-TB results had comparable sensitivity (82·9% and 84·4%) to respiratory culture and Xpert for confirmed TB, and robust sensitivity (80·5% and 76·5%) for unconfirmed TB, with differential specificity for unlikely TB cases with and without TB consistent symptoms (78·4–98·1%). CFP10pep levels decreased by six months post-treatment initiation only in children with positive treatment responses. Interpretation Serum CFP10pep detection can effectively diagnose pediatric TB, including unconfirmed and extrapulmonary TB cases missed by sputum-based methods, while decreases after treatment anti-TB treatment initiation can monitoring effective treatment responses.