Protein copy numbers can be measured by biochemical methods ranging from quantitative Western Blotting to several mass spectrometry approaches. Such methods only provide average copy numbers, obtained over large cell numbers. However, copy number estimates for single cells or single organelles could be obtained by combining biochemical characterizations with an imaging approach. We performed this here for synaptic proteins, in a protocol that we termed comparative synaptosome imaging for semi-quantitative copy numbers (CosiQuant). In brief, in CosiQuant we immunostain in parallel biochemically-characterized synaptosomes, for which we have already determined the average protein copy numbers, and the samples of interest (such as neuronal cultures). We then derive the copy numbers in the samples of interest by comparing the immunofluorescence intensities. We measured the intensities not only in arbitrary fluorescence units, but also as numbers of antibodies per synaptosome, for a large number of targets. This implies that other groups can immediately apply CosiQuant for these targets, by simply estimating the number of antibodies per structure of interest. CosiQuant should therefore be a useful addition to the growing set of imaging techniques for synaptic neuroscience.