Abstract We present a lensfree imaging method to analyze polarity in RWPE1 prostate epithelial cells that form polarized acini with lumen under standard tridimensional (3D) culture conditions. The first event in epithelial carcinogenesis is loss of polarity, followed by uncontrolled proliferation leading to metastasis. We demonstrate that it is possible to use optical signatures to discriminate 3D objects with distinct polarities in a large field of view. The three metrics we present here are designed as image processing tools to discriminate acini from spheroids without any 3D reconstruction. To demonstrate that our lensfree imaging platform may be used to study the 3D organization of epithelial cells, we analyzed and quantified the modulation of dynamic processes, e.g., the polarity of acini and the merging of polarized structures, upon transforming growth factor beta-1 (TGF beta-1) addition to the culture media. Hence, coupling lensfree microscopy with 3D cell culture provides an innovative tool to study epithelial tissue morphogenesis in a large field of view and to elucidate the regulation of growth, morphogenesis and differentiation in normal and cancerous human prostate cells. Moreover, such biosensor would be a powerful tool to follow cancer progression and to evaluate anti-cancer drugs.