AbstractTraditional manual gating strategies are often time-intensive, place a high burden on the analyzer, and are susceptible to bias between analyzers. Several automated gating methods have shown to exceed performance of manual gating for a limited number of cell subsets. However, many of the automated algorithms still require significant manual interventions or have yet to demonstrate their utility in large datasets. Therefore, we developed an approach that utilizes a previously published automated algorithm (OpenCyto framework) with a manually created hierarchically cell gating template implemented, along with a custom developed visualization software (FlowAnnotator) to rapidly and efficiently analyze immunophenotyping data in large population studies. This approach allows pre-defining populations that can be analyzed solely by automated analysis and incorporating manual refinement for smaller downstream populations. We validated this method with traditional manual gating strategies for 24 subsets of T cells, B cells, NK cells, monocytes and dendritic cells in 931 participants from the Health and Retirement Study (HRS). Our results show a high degree of correlation (r ≥ 0.80) for 18 (78%) of the 24 cell subsets. For the remaining subsets, the correlation was low (<0.80) primarily because of the low numbers of events recorded in these subsets. The mean difference in the absolute counts between the hybrid method and manual gating strategy of these cell subsets showed results that were very similar to the traditional manual gating method. We describe a practical method for standardization of immunophenotyping methods in large scale population studies that provides a rapid, accurate and reproducible alternative to labor intensive manual gating strategies.