Droplet Digital PCR (ddPCR™) enables accurate, precise, and sensitive quantification of specific nucleic acid sequences. In addition to the standard detection of two targets using two different fluorophores, it is possible to increase the number of targets detected by varying parameters that affect PCR efficiency and end-point fluorescence. In this case, we describe a method to multiplex assays by varying the concentrations of primers and probes or the type of fluorophores used. This allows users to expand the number of simultaneously detected targets up to four. Increasing the number of potential targets per test is a significant improvement for ddPCR, dramatically augmenting the information output of each sample.