Image-based high-throughput screening (HTS) reveals a high level of heterogeneity in single cells and multiple cellular states may be observed within a single population. Currently available high-dimensional analysis methods are successful in characterizing cellular heterogeneity, but suffer from the “curse of dimensionality” and non-standardized outputs. Here we introduce RefCell, a multi-dimensional analysis pipeline for image-based HTS that reproducibly captures cells with typical combinations of features in reference states and uses these “typical cells” as a reference for classification and weighting of metrics. RefCell quantitatively assesses heterogeneous deviations from typical behavior for each analyzed perturbation or sample. We apply RefCell to the analysis of data from a high-throughput imaging screen of a library of 320 ubiquitin-targeted siRNAs selected to gain insights into the mechanisms of premature aging (progeria). RefCell yields results comparable to a more complex clustering-based single-cell analysis method; both methods reveal more potential hits than a conventional analysis based on averages.