Plasmodium falciparum genotyping has recently undergone a revolution, and genome-wide genotype datasets are now being collected for large numbers of parasite isolates. By contrast, phenotyping technologies have lagged behind, with few high throughput phenotyping platforms available. Invasion of human erythrocytes by Plasmodium falciparum is a phenotype of particular interest because of its central role in parasite development. Invasion is a variable phenotype influenced by natural genetic variation in both the parasite and host and is governed by multiple overlapping and in some instances redundant parasite-erythrocyte interactions. To facilitate the scale-up of erythrocyte invasion phenotyping, we have developed a novel platform based on two-color flow cytometry that distinguishes parasite invasion from parasite growth. Target cells that had one or more receptors removed using enzymatic treatment were prelabeled with intracellular dyes CFDA-SE or DDAO-SE, incubated with P. falciparum parasites, and parasites that had invaded either labeled or unlabeled cells were detected with fluorescent DNA-intercalating dyes Hoechst 33342 or SYBR Green I. Neither cell label interfered with erythrocyte invasion, and the combination of cell and parasite dyes recapitulated known invasion phenotypes for three standard laboratory strains. Three different dye combinations with minimal overlap have been validated, meaning the same assay can be adapted to instruments harboring several different combinations of laser lines. The assay is sensitive, operates in a 96-well format, and can be used to quantitate the impact of natural or experimental genetic variation on erythrocyte invasion efficiency.