Pharmaceutical industries have declared their need of metrology in the cellular field, to improve new drugs developing time and costs by high-content screening technologies. Cell viability and proliferation tests largely use confluency of cells on a bi-dimensional (2D) surface as a biological measurand. The confluency is measured from images of 2D surface acquired via microscopy techniques. The plethora of algorithms already in use aims at recognizing objects from images and identifies a threshold to distinguish objects from the background. The reference method is the visual assessment from an operator and any objective uncertainty estimation is not yet available. A method to estimate the image analysis contribution to confluency uncertainty is here proposed. A maximum and a minimum threshold are identified from a visual assessment of the free edge of the cells. An application to a fluorescence microscopy image of 2D of PT-45 cell cultures is reported. Results shows that the method can be a promising solution to associate an uncertainty to cell confluency measurements to enhance reliability and efficiency of high-content screening technologies.