BACKGROUND Omalizumab is effectively used in asthma therapy, but ELISA methods used for omalizumab determination in blood from asthma patients may be interfered by the pre-existing IgE. OBJECTIVE To reduce the effect of pre-existing IgE on the omalizumab determination, the authors proposed a novel ELISA that can eliminate pre-existing IgE with by acid dissociation. METHODS The method was developed by dissociating the IgE-omalizumab complex with glacial acetic acid, and bio-IgE was added to bind the free omalizumab in serum, then bio-IgE-complex was captured by the immobilized streptavidin and detected by HRP-conjugated mouse anti-human IgG. Then a full validation of standard curve fitness, precision, accuracy, dilutional linearity, specificity, selectivity, stability, hook effect, and parallelism was performed. At last, the method was used in two studies in compliance with Good Laboratory Practice. RESULTS Correlation coefficient R2 obtained from each calibration curve was 0.999 or 1.000 in the detection range of 0.1 μg/mL to 12.8 μg/mL. Results of precision, accuracy, dilutional linearity, specificity, selectivity, stability, hook effect, and parallelism were acceptable according to the ICH guideline M10. The method was successfully used in omalizumab determination in serum from 20 monkeys treated with 150 mg/kg omalizumab. CONCLUSIONS In conclusion, by dissociating IgE-omalizumab complex, the authors proposed for the first time a new validated IgE-tolerant ELISA assay to determine omalizumab concentration in serum samples.