Background <p>Differentiating STEC-HUS from aHUS can be challenging due to overlapping clinical presentations. Confirming STEC infection is crucial to guide treatment. Although fecal testing is the gold standard, it confirms the diagnosis in only about half of HUS cases. Detection of anti-O-polysaccharide antibodies (anti-LPS) by using Glyco-iELISA can improve the diagnosis rate. The aim of this study was to assess the diagnostic contribution of anti-LPS for STEC confirmation in patients with negative stool results with consideration of sample collection time, the presence/absence of bloody diarrhea, and prior antibiotic use.</p> Methods <p>A two-center retrospective study was conducted, including 137 patients with STEC-HUS evaluated by both fecal PCR and anti-LPS testing.</p> Results <p>STEC infection was confirmed in 45 (35.2%) patients by stool culture, in 87 (63.5%) by PCR, and in 100 (73%) by anti-LPS. Anti-LPS was positive in 35 patients with negative stool results, increasing the diagnostic rate to 90.5%, with an added diagnostic value of 25.5% (<i>p</i> = 0.0001). Stool STEC detection was significantly lower in patients without bloody diarrhea (<i>p</i> = 0.02), in those who received antibiotics (<i>p</i> = 0.004), and in those whose stool samples were collected ≥ 7 days after symptom onset (<i>p</i> = 0.0002). After multivariate analysis, only the use of antibiotics (<i>p</i> = 0.01) and delayed sample collection (<i>p</i> = 0.002) remained negatively associated with stool STEC detection. In contrast, anti-LPS antibodies’ results were unaffected by these variables.</p> Conclusions <p>STEC infection was confirmed in 90.5% of children with STEC-HUS. Anti-LPS serology significantly contributed to diagnosis, especially in patients with delayed stool tests and in those treated with antibiotics.</p> Graphical abstract <p></p>