BACKGROUND Biomedical image processing methods require users to optimise input parameters to ensure high-quality output. This presents two challenges. First, it is difficult to optimise multiple input parameters for multiple input images. Second, it is difficult to achieve an understanding of underlying algorithms, in particular, relationships between input and output. RESULTS We present a visualisation method that transforms users' ability to understand algorithm behaviour by integrating input and output, and by supporting exploration of their relationships. We discuss its application to a colour deconvolution technique for stained histology images and show how it enabled a domain expert to identify suitable parameter values for the deconvolution of two types of images, and metrics to quantify deconvolution performance. It also enabled a breakthrough in understanding by invalidating an underlying assumption about the algorithm. CONCLUSIONS The visualisation method presented here provides analysis capability for multiple inputs and outputs in biomedical image processing that is not supported by previous analysis software. The analysis supported by our method is not feasible with conventional trial-and-error approaches.