Conventional 3D culture is typically performed in multi-well plates (e.g. 12 wells). The volumes and dimensions necessitate relatively large numbers of cells and fluid exchange steps are not easily automated limiting throughput. 3D microchannel culture can overcome these challenges simplifying 3D culture processes. However, the adaptation of immunocytochemical endpoint measurements and the validation of microchannel 3D culture with conventional 3D culture are needed before widespread adoption can occur. Here we use a breast carcinoma growth model governed by complex and reciprocal interactions between epithelial carcinoma cells and mesenchymal fibroblasts to validate the 3D microculture system. Specifically, we report the use of a 3D microchannel co-culture assay platform to interrogate paracrine signalling pathways in breast cancer. Using a previously validated 3D co-culture of human mammary fibroblasts and T47D breast carcinoma cells, we demonstrate the use of arrayed microchannels to analyze paracrine signalling pathways and screen for inhibitors. Results in both conventional format (multiwell plate) and microchannels were comparable. This technology represents a significant advancement for high-throughput screening in individual patients and for drug discovery by enabling the use of 3D co-culture models via smaller sample requirements and compatibility with existing HTS infrastructure (e.g. automated liquid handlers, scanners).