HER2 amplification and/or overexpression in breast cancer are adverse prognostic factors and can predict the response to trastuzumab therapy. As assessment of HER2 status in breast cancer is of great importance for clinical decision-making, it is crucial to have reliable methods to quantify HER2. In the present project, we have developed a modification of the direct-double-differential PCR method (dddPCR) for gene dosage quantification of HER2 in breast cancer samples. The study was also undertaken to establish the potential application of the newly developed dddPCR method for HER2 testing in a diagnostic laboratory. The dddPCR validity for determining HER2 amplification in comparison to IHC-based HercepTest for the detection of protein levels was performed. Modified dddPCR was proven to be highly reproducible and reliable. A statistically significant correlation between protein expression and gene dosage of HER2 was found in the examined material. The obtained results indicate that dddPCR provides a fast and reliable diagnostic tool for HER2 quantification and may be considered as a supplementary method to verify equivocal results obtained with IHC-based HercepTest. Notably, dddPCR can be performed in every basic molecular biology laboratory, and is inexpensive and time-saving.