Background: Polarization of tissue is achieved by asymmetric distribution of proteins and organelles within individual cells. However, existing quantitative assays to measure this asymmetry in an automated and unbiased manner suffer from significant limitations. Results: Here, we report a new way to assess protein and organelle localization in tissue based on correlative fluorescence analysis. As a proof of principle, we successfully characterized planar cell polarity dependent asymmetry in developing Drosophila melanogaster tissues on the single cell level using fluorescence cross-correlation. Conclusions: Systematic modulation of signal strength and distribution show that fluorescence cross-correlation reliably detects asymmetry over a broad parameter space. The novel method described here produces robust, rapid, and unbiased measurement of biometrical properties of cell components in live tissue that is readily applicable in other model systems. Developmental Dynamics 241:1301–1309, 2012. © 2012 Wiley Periodicals, Inc.