Background: ESR1 mutations are a leading cause of acquired resistance to endocrine therapy in HR+/HER2- metastatic breast cancer (mBC). Monitoring of ESR1 mutations' dynamic accumulations is critical for patients to quickly switch to effective selective estrogen receptor degrader (SERD) treatment. Therefore, a rapid and cost-effective assay for ESR1 mutations status monitoring is in urgent demand in clinical practice. Methods: ESR1 sequence with consecutive mutations poses a major hurdle for conventional qPCR assay development. Here an Innovative Suppression Probe PCR was designed and assay was successfully developed (Eq-PCR). Genomic cell-free DNA samples which were extracted from ESR1 mutated breast cancer cell lines (n=47) and patient-derived peripheral blood samples (n=25) were subjected to Eq-PCR, ddPCR (digital droplet PCR) and Next generation sequencing (NGS covering 78 genes). Results: Our Eq-PCR assay has a limit of detection of 0.5% mutation frequency for major ESR1 mutations. For 47 cell lines samples (10ng DNA input), overall agreement between Eq-PCR and ddPCR is 100%. For 25 samples collected from mBC patients(cfDNA concentration ranged from 0.2ng-0.8ng/μl), overall agreement between Eq-PCR and NGS is 100.0% of PPV (5 positive samples) and 85.0% of NPV (20 negative samples). Conclusion: A novel, cost-effective qPCR-based assay for major ESR1 mutations was developed with top accuracy and quick turn-around time. This assay provides an urgently needed clinical tool to monitor critical ESR1 mutation in HR+/HER2- mBC patients in a convenient, timely and cost-effective fashion.