Background Laboratory diagnosis of Lyme disease (LD) relies on a two-tier protocol. We have observed disproportionate equivocal serologies in children requiring reflex western blot (WB) using manufacturer-provided ranges based on adult studies. We aimed to determine appropriate ranges for our pediatric population. Methods We performed a one-year retrospective institutional review of all 2755 children with LD testing with the Vidas® Lyme IgM II/IgG II immunoassays with reflex to WB for equivocal/positive serologies. Results were assessed by frequency distributions, optimization via percent agreement analysis, and clinical adjudication. Results The proposed ranges for IgM (negative ≤0.20, equivocal ≥0.21 to 0.50) allowed for a decrease in the IgM equivocal rate (7% to 2%) and IgG positive rate (15% to 13%). There was a decrease in the positive percent agreement between tiers (95% to 83% and 98% to 95%) with increase in the negative (32% to 63% and 70% to 81%) and overall (65% to 73% and 85% to 88%) percent agreements for IgM and IgG, respectively. Of 15 IgM serologies reclassified as negative with a positive WB and not positive for IgG, 8 were clinically negative, 5 were clinically positive, and two had insufficient history. Of the 10 IgG serologies reclassified as negative with a positive WB 3 were clinically positive, 6 were clinically negative and one had insufficient history. Conclusions Our modified ranges are more suitable for our pediatric population while reducing overdiagnosis, unnecessary treatment, diagnostic uncertainty, and turnaround time.