The PI3K/AKT pathway is a critical downstream signal from HER2, and its alterations are potential biomarkers for predicting the effectiveness of HER2-targeted therapy. Understanding the regulation of this pathway can contribute to breast cancer management. The recent increase in the clinical use of PI3K/AKT inhibitors for breast cancer requires characterizing patient populations at both clinical and molecular levels to provide more appropriate and personalized treatments. In previous studies using immunohistochemistry we observed the activation state of PI3K/AKT pathway in HER2+ and triple-negative breast cancer patients compared to the luminal subtype. Since biomarkers from liquid biopsies offer the advantage of non-invasively clinical monitoring, we aimed to identify circulating miRNAs associated with the PI3K/AKT pathway specifically in HER2+ patients who undergo neoadjuvant treatment. Biobanks allow the evaluation of a larger number of samples; however, most plasma samples are usually obtained with heparin, which has an inhibitory effect on RNA and DNA polymerase. Thus, we recognized the need to develop a protocol to counteract heparin interference. To address this, we evaluated different options to improve miRNAs extraction protocols using carriers, such as glycogen and tRNA, and heparin inhibitors, like LiCl and protamine sulfate. We applied these optimized protocols to samples from five HER2+ breast cancer patients who received trastuzumab plus pertuzumab combined with chemotherapy before surgery. We verified the effectiveness of our protocols by amplifying miR16-5p, -155-5p, -195-5p, -21-5p, and -126- 5p using RT-qPCR in pre- and post-therapy heparinized plasma samples. We are now validating this set of miRNAs in groups of anti-HER2 therapy responding and non-responding patients. This approach could be highly beneficial for monitoring and predicting responses to specific antitumor treatments.