Chromogenic in situ hybridization (CISH) is a common technique that is used for human epidermal growth factor receptor 2 (HER2) assessment in breast cancer diagnosis. CISH is based on the enzymatic amplification of DNA markers, generating a colorimetric image that can be visualized by a bright-field microscope and does not require a fluorescence detection setup. However, CISH is still characterized by a laborious and lengthy staining process, and is expensive due to the cost of the DNA probe solution. In this paper, we present a new technique named microfluidic-assisted chromogenic in situ hybridization (MA-CISH) in which hydrodynamic flow patterns in a microfluidic chip are used to improve hybridization efficiency between probe and target, thereby reducing the hybridization time and the consumption of probe solution. We optimized the MA-CISH protocol with a fractional factorial design of experiments using a SKBR3 breast cancer cell line. Optimized MA-CISH exploits discontinuous flow of DNA probe solution, enabling the reduction of the hybridization time from overnight down to 2 h and requiring 70% less probe solution, while achieving a better signal quality than the standard CISH technique. Optimized MA-CISH was successfully tested for several breast cancer tissue samples, showing a good correlation with standard CISH results as evaluated on adjacent slides. Finally, an automatic scoring software was developed to facilitate CISH signal evaluation, and showed good agreement with the traditional counting protocol.