The application of multiplexed imaging technologies in phenotypic drug discovery (PDD) enables profiling of complex cellular perturbations in response to drug treatment. High-content analysis (HCA) is among the most pursued approaches in PDD, with a proven capability to identify compounds with a given cellular mechanism of action (MOA), as well as to unveil unexpected drug cellular activities. The ability of fluorescent image-based cytometric techniques to dissect the phenotypic heterogeneity of cell populations depends on the degree of multiplexing achievable. At present, most high-content assays employ up to four cellular markers separately detected in distinct fluorescence channels. We explored the possibility to increase HCA multiplexing through analysis of multiple proliferation markers in the same fluorescence channel by taking advantage of the different timing of antigen appearance during the cell cycle, or differential intracellular localization. Simultaneous analysis of DAPI staining and five immunofluorescence markers (BrdU incorporation, active caspase-3, phospho-histone H3, phospho-S6, and Ki-67) resulted in the first six-marker high-content assay readily applicable to compound MOA studies. This approach allows detection of rare cell subpopulations, unveiling a high degree of phenotypic heterogeneity in exponentially growing cell cultures and variability in the individual cell response to antiproliferative drugs.