In vitro opsonophagocytosis killing assay (OPA) is widely accepted to quantitate Streptococcus pneumococcal antibodies to serotype-specific pneumococcal capsular polysaccharide (PS). A titer estimation method is needed for large scale data generated by OPA, and it is one component of OPA standardization. In order to improve the reliability of OPA results, we developed a nonlinear fitting method using the Levenberg–Marquardt algorithm with an option of a robust procedure to estimate titers of OPA data. Performance of the proposed method was evaluated by comparing precision and accuracy of titer estimation with traditional methods used in the literature by analyzing real experimental data sets. Goodness-of-fit to experimental data for the two model-based methods was also assessed. We conclude that the four-parameter logistic model is an alternative choice for titer estimation of OPA data. Computer software using the statistical language R and Microsoft Excel was developed to implement our calculation algorithm for OPA data.