Streptococcus pneumoniae (the pneumococcus) is a leading cause of paediatric mortality worldwide. Nasopharyngeal (NP) carriage is considered a prerequisite for pneumococcal disease. Often more than one serotype is present, and multiple serotype carriage (MSC) underpins serotype replacement, a key risk to current global immunization programs, especially in developing countries. Currently, there is no standard laboratory method to detect MSC. A variety of new methods show promise, but lack data describing and comparing their performance. We developed a set of reference samples comprising 81 spiked samples (containing 0-4 serotypes in varying amounts) and 260 NP samples from children in developing countries. Twenty groups developing new serotyping methods tested the spiked samples, and the sensitivity and positive predictive values (PPV, true positives) were determined. Methods were evaluated based upon performance and other technical properties. Some methods were unsuitable for detecting MSC (overall sensitivity < 70% and/or PPV < 90%). Others performed well but had technical limitations. Four methods, based on real-time PCR, microarray, latex agglutination and tRFLP technologies, had sensitivities to detect the major serotype populations of 99%, 100%, 92%, and 96%; sensitivities to detect the minor serotype populations of 99%, 95%, 78% and 87%; and PPV of 99%, 100%, 93% and 99% respectively using the spiked samples and were selected to test the 260 field samples. Preliminary data suggest similar sensitivity and PPV as for the spiked samples, although discriminating between false and true positives remains a challenge. To assist with this, a single-plex quantitative PCR assay for all serotypes is being developed. From these results, the PneuCarriage project will identify the optimal method(s) to detect MSC according to set criteria including scientific performance and applicability to resource-poor settings.