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Mosaic analysis provides a means to probe developmental processes in situ by generating loss-of-function mutants within otherwise wildtype tissues. Combining these techniques with quantitative microscopy enables researchers to rigorously compare RNA or protein expression across the resultant clones. However, visual inspection of mosaic tissues remains common in the literature because quantification demands considerable labor and computational expertise. Practitioners must segment cell membranes or cell nuclei from a tissue and annotate the clones before their data are suitable for analysis. Here, we introduce Fly-QMA, a computational framework that automates each of these tasks for confocal microscopy images of Drosophila imaginal discs. The framework includes an unsupervised annotation algorithm that incorporates spatial context to inform the genetic identity of each cell. We use a combination of real and synthetic validation data to survey the performance of the annotation algorithm across a broad range of conditions. By contributing our framework to the open-source software ecosystem, we aim to contribute to the current move toward automated quantitative analysis among developmental biologists. Author summary: Biologists use mosaic tissues to compare the behavior of genetically distinct cells within an otherwise equivalent context. The ensuing analysis is often limited to qualitative insight. However, it is becoming clear that quantitative models are needed to unravel the complexities of many biological systems. In this manuscript we introduce a computational framework that automates the quantification of mosaic analysis for Drosophila imaginal discs, a common setting for studies of developmental processes. The software extracts quantitative measurements from confocal images of mosaic tissues, rectifies any cross-talk between fluorescent reporters, and identifies clonally-related subpopulations of cells. Together, these functions allow users to rigorously ascribe changes in gene expression to the presence or absence of particular genes. We validate the performance of our framework using both real and synthetic data. We invite interested readers to apply these methods using our freely available software. [ABSTRACT FROM AUTHOR]