BACKGROUND: The diagnosis of septic arthritis (SA) and osteomyelitis (OM) has remained challenging in the pediatric population, often accompanied by delays and requiring invasive interventions. The purpose of this pilot study is to identify a novel panel of biomarkers and cytokines that can accurately differentiate SA and OM at initial presentation using serum alone. METHODS: Twenty patients below 18 years old whose working diagnosis included SA (n=10) and OM (n=10) were identified. Serum was collected at initial evaluation. Each sample underwent seven ELISA [C1-C2, COMP, CS-846, hyaluronan, procalcitonin, PIIANP, C-terminal telopeptide of type II collagen (CTX-II)] and 65-plex cytokine panels. Principal component and Lasso regression analysis were performed to identify a limited set of predictive biomarkers. RESULTS: Mean age was 4.7 and 9.5 years in SA and OM patients, respectively (P=0.029). 50% of SA patients presented within 24 hours of symptom onset, compared with 0% of OM patients (P=0.033). 30% of SA patients were discharged home with an incorrect diagnosis and re-presented to the emergency department days later. At time of presentation: temperature ≥38.5°C was present in 10% of SA and 40% of OM patients (P=0.12), mean erythrocyte sedimentation rate (mm/h) was 51.6 in SA and 44.9 in OM patients (P=0.63), mean C-reactive protein (mg/dL) was 55.8 in SA and 71.8 in OM patients (P=0.53), and mean white blood cells (K/mm3) was 12.5 in SA and 10.4 in OM patients (P=0.34). 90% of SA patients presented with ≤2 of the Kocher criteria. 100% of SA and 40% of OM patients underwent surgery. 70% of SA cultures were culture negative, 10% MSSA, 10% Kingella, and 10% Strep pyogenes. 40% of OM cultures were culture negative, 50% MSSA, and 10% MRSA. Four biomarkers [CTx-II, transforming growth factor alpha (TGF-α), monocyte chemoattractant protein 1 (MCP-1), B cell-attracting chemokine 1] were identified that were able to classify and differentiate 18 of the 20 SA and OM cases correctly, with 90% sensitivity and 80% specificity. CONCLUSIONS: This pilot study identifies a panel of biomarkers that can differentiate between SA and OM at initial presentation using serum alone. LEVEL OF EVIDENCE: Level II-diagnostic study.