The objective of this study was to develop a new real time PCR-based method for quantitative detection of topoisomerase II alpha ( TOP2A) aberrations and to evaluate its clinical utility in breast cancer. The method applied dually labelled hydrolysis probes and Pfaffl quantification method. The study group consisted of 83 consecutive breast cancer patients. In the examined tumour samples median TOP2A gene dosage was 1.08 (range 0.34–7.55). TOP2A amplifications were found in 12 tumours (14.5%), no deletion was detected. Statistically significant positive correlation of TOP2A gene dosage with nodal status, tumour grade, and HER2 protein status was found. TOP2A status also correlated with disease free survival. The newly developed real time PCR assay showed to be fast and easy to perform. Determined by the method TOP2A gene dosage was shown to be a potent prognostic factor in breast cancer.