Advancements in technologies for assessing biomechanics at the cellular level have led to discoveries in mechanotransduction and the investigation of cell mechanics as a biomarker for disease. With the recent development of an integrated optical tweezer with micron resolution particle image velocimetry, the opportunity to apply controlled multiaxial stresses to suspended single cells is available (Nève, N., Lingwood, J. K., Zimmerman, J., Kohles, S. S., and Tretheway, D. C., 2008, “The : An Integrated Particle Image Velocimetry and Optical Tweezers Instrument for Microenvironment Investigations,” Meas. Sci. Technol., 19(9), pp. 095403). A stress analysis was applied to experimental and theoretical flow velocity gradients of suspended cell-sized polystyrene microspheres demonstrating the relevant geometry of nonadhered spherical cells, as observed for osteoblasts, chondrocytes, and fibroblasts. Three flow conditions were assessed: a uniform flow field generated by moving the fluid sample with an automated translation stage, a gravity driven flow through a straight microchannel, and a gravity driven flow through a microchannel cross junction. The analysis showed that fluid-induced stresses on suspended cells (hydrodynamic shear, normal, and principal stresses in the range of 0.02–0.04 Pa) are generally at least an order of magnitude lower than adhered single cell studies for uniform and straight microchannel flows (0.5–1.0 Pa). In addition, hydrostatic pressures dominate (1–100 Pa) over hydrodynamic stresses. However, in a cross junction configuration, orders of magnitude larger hydrodynamic stresses are possible without the influence of physical contact and with minimal laser trapping power.