We developed fluorescent mono- and multivalent opsonophagocytic assays (fOPA and fmOPA, respectively) specific for seven Streptococcus pneumoniae serotypes (4, 6B, 9V, 14, 18C, 19F, and 23F). Bacterial survival was quantitated with alamar blue, a fluorescent metabolic indicator. Both fOPA and fmOPA allow for determination of viability endpoints for up to seven serotypes with high levels of agreement to the reference method. The fmOPA eliminates colony counting, reduces serum volume, and produces results in 1 day.