Despite the significance of cellular morphology as a functional phenotype, it remains challenging to quantitatively relate morphological phenotype to the behavior of subcellular molecules. Molecular studies have identified many components controlling cell morphogenesis, but it is unclear how this information is translated into the physical world. In plant cells, growth requires synthesis of cytoplasmic components as well as expansion of the cell wall. The cell wall is a stiff yet flexible polymeric network that encapsulates cells and counterbalances stress created by turgor pressure inside the cell, thereby controlling cell shape. It is now well established that the cytoskeleton plays a key role in the biogenesis and morphogenesis of the cell wall. While the microtubules guide cellulose synthase complex movement (Paredez et al. 2006), the actin network is responsible for global distribution of cellulose synthase complexes (Gutierrez et al. 2009; Sampathkumar et al. 2013). It is also suggested that mechanical stresses orient the microtubules along their principal direction (Hamant et al. 2008; Sampathkumar, Krupinski, et al. 2014; Sampathkumar, Yan, et al. 2014). Nevertheless, to fully understand how plant cells are shaped and how external mechanical stresses influence this process, a quantitative approach to evaluate the mechano-response in single cells needs to be established. Here we present a technique (Chang et al. 2014) to confine single plant protoplasts into molds of defined shapes. The principle is to confine a plant protoplast expressing fluorescent cytoskeletal reporters into micro-wells of different shapes with sizes of 10 to 30 μm. The protoplasts are then monitored with a confocal microscope to evaluate changes in cytoskeletal organization and dynamics during the process of symmetry breaking. These experiments are the basis of assessing quantitatively how different shapes control cytoskeleton organization behavior by regulating the distribution of physical stresses.