A protocol is described which allows the quantification of cells belonging to one type within a coculture of two cell types. For this, a series of micrographs recorded under different conditions (transmission, different fluorescence wavelength) were processed using an image recognition software. To show the effectiveness of the protocol, the relative amounts of adherent cells in three different cocultures of two cell types were determined as a function of the chemical patterning of substrates. (C) 2010 Elsevier Ltd. All rights reserved.