Abstract: The present invention relates to an in vitro method for identifying and/or characterizing one or more mutations in a hotspot mutation sequence of at least one ESR1 target fragment from a DNA sample, said method comprising subjecting the DNA sample to a drop-off digital polymerase chain reaction (PCR) in the presence of a PCR solution comprising: a pair of primers suitable for amplifying an ESR1 target fragment; an oligonucleotide reference (REF) hydrolysis probe, labeled with a fluorophore, wherein said REF oligonucleotide probe is complementary to a wild-type sequence of the target fragment located outside of the hotspot mutation sequence; an oligonucleotide hotspot (HOTSPOT) hydrolysis probe, labeled with another fluorophore, wherein said oligonucleotide HOTSPOT probe is complementary to a wild-type sequence of the hotspot mutation sequence of the target DNA fragment.