Sequential chromatin immunoprecipitation (SeqChIP) is a procedure in which formaldehyde-crosslinked, protein-DNA complexes from living cells are subjected to two sequential immunoprecipitations with antibodies of different specificity. SeqChIP has been used to address, in a qualitative manner, whether two proteins can simultaneously co-occupy a stretch of DNA in vivo. Here, we expand on our earlier work and describe theoretical and practical considerations for performing and interpreting SeqChIP experiments in a quantitative manner. We provide a detailed experimental procedure for designing and performing SeqChIP experiments as well as experimental examples of the three possible outcomes: full co-occupancy, no co-occupancy and partial co-occupancy. In some cases of partial co-occupancy, the order of immunoprecipitations in SeqChIP can strongly influence the outcome. We experimentally confirm a quantitative parameter that provides a measure of co-occupancy of two proteins on a given region of DNA and provide information on how to interpret the results of SeqChIP experiments. Our quantitative treatment of SeqChIP data substantially expands the usefulness of the technique for elucidating molecular mechanisms in vivo.