HisTOOLogy is a free software tool for extracting useful numerical information from colour images of histological sections. The software's simple Graphical User Interface (GUI) is easy to use, even for non-experts. It is based on an innovative colour separation method which groups colours such that colour pixels in the same group (called a cluster) are similar to each other and different from those in other groups (clusters). The clustering method accurately and reproducibly allows the quantification of different tissue components. This paper describes HisTOOLogy's main features and GUI structure, showing how it can be used to separate dye components in different stains. In addition, to demonstrate how the tool can be employed to obtain quantitative information on biological tissues, the effect of different hepatic tissue decellularization protocols on cell removal and matrix preservation was assessed using HisTOOLogy and compared with conventional DNA and total protein content assays. HisTOOLogy's performance was also compared with ImageJ to demonstrate its advantages terms of ease of use and speed of colour separation. The software is available from this link: www.centropiaggio.unipi.it/content/histoology-download.