A major obstacle in ovarian cancer treatment is the onset of ascites, an abnormal build-up of fluid in the peritoneal cavity. Using in vitro perfusion models, ascitic flow has been shown to drive epithelial-mesenchymal-transition (EMT) biomarker expression, promote epidermal growth factor receptor (EGFR) downstream signaling, and upregulate chemoresistance. Given the close ties between cell mechanics and behaviors, it is of interest to establish if mechanotransduction serves a role in cell signaling dysfunction. Here, we identified the mechanical behavior of tumor spheroids subjected to flow using Brillouin confocal microscopy, a non-contact optical method based on the interaction between incident light and microscopic mechanical waves within matter. We validated this technique by establishing a relationship with the traditionally derived Young’s modulus measured using atomic force microscopy and a parallel-plate compression device. Following characterization, we used Brillouin confocal microscopy to map mechanical properties of tumor spheroids embedded in a microfluidic chip and found that continuous flow for 7 days caused a decreased Brillouin shift (i.e., stiffness) compared to tumors in a static condition. Another physical phenomenon related to ascites is dysregulated osmolality. Maintaining cell water homeostasis is driven by the transport of water to balance solute concentration and can have direct consequences on mechanics and biochemical signaling in cells. Recently, it was demonstrated in single cells that cell volume correlated with mechanical properties; but the effects in tumor spheroids which exhibit multi-cellular interfaces has remained unclear. Here, we derived relationships between osmolality and nuclear volume, tumor cell density, and Young’s modulus, and found the correlations in spheroids resembled single cell relationships previously described in literature. Additionally, we looked at the impact of osmotic shocks on E-cadherin junctions and found aggregates formed with a unique timescale compared to morphology. Lastly, we observed reversibility of the mechanical, morphological, and molecular properties which showed the tumor’s dynamic ability to respond to physical cues. Altogether, this work demonstrated how flow and osmosis associated with ovarian cancer ascites can trigger phenotype transformations. These findings warrant future investigations into how the regulation of mechanotransduction pathways can be harnessed to prevent chemoresistance and signaling dysfunction.