Opsonization assay has become an important tool in assessing pneumococcal vaccine immunogenicity (reference 1). To facilitate the widespread use of the opsonization assay, we have prepared a detailed protocol for 4-fold multiplexed opsonophagocytic killing assay (UAB-MOPA). Although this protocol describes the multiplexed assay, this protocol can be used as a single serotype assay with the minor changes described in Note 9 in Assay Notes section. Also, this protocol can be used with minor alterations (e.g., a different serial dilution factor or different number of wells per sample). Additional information on this assay can be found in publications (references 2 and 3) and at a web site (www.vaccine.uab.edu).