•Sensitive and robust multiplex dPCR assays for the simultaneous detection of 11 ESR1mutations.•At least one mutation for 28.4% of the 109 HR+/HER2- Metastatic Breast Cancer patients tested.•Cross-validation with the multiplex dPCR assay used for the PADA-1 study, with 91% concordance.•ESR1mutation detection is significantly associated with liver metastases (p = 0.0091).•Very suited for ESR1mutations monitoring in the plasma of Metastatic Breast Cancer patients. Early detection of ESR1 mutations is a key element for better personalization of the management of patients with HR+/HER2- Metastatic Breast Cancer (MBC). Analysis of circulating tumor DNA from liquid biopsies is a particularly well-suited strategy for longitudinal monitoring of such patients. Using the naica® three-color digital PCR platform, we developed a screening assay allowing the detection of 11 ESR1 mutations and designed a sequential strategy for precise mutation identification. We then applied this strategy in the analysis of plasma circulating cell-free DNA from 109 HR+/HER2- MBC patients and performed a double-blind comparison study on a subset of patients with the multiplex assay used at the Institut Curie (IC) for the PADA-1 study. Thirty-one patients (28.4%) harboured at least one ESR1 mutation, with the following frequencies: D538G (41.03%), Y537S (25.64%), E380Q (10.26%), Y537N (10.26%), “(536–540)” (7.69%), Y537C (2.56%), and L536R (2.56%). The presence of ESR1 mutation(s) was significantly associated with liver metastases (p = 0.0091). A very good agreement (91%) was observed with the IC assay. Our assays have proven to be robust and highly sensitive and are very well-suited for monitoring ESR1 mutations in the plasma of MBC patients.