The orientation of cells and associated F-actin stress fibers is essential for proper tissue functioning. We have previously developed a computational model that qualitatively describes stress fiber orientation in response to a range of mechanical stimuli. In this paper, the aim is to quantitatively validate the model in a static, heterogeneous environment. The stress fiber orientation in uniaxially and biaxially constrained microscale tissues was investigated using a recently developed experimental system. Computed and experimental stress fiber orientations were compared, while accounting for changes in orientation with location in the tissue. This allowed for validation of the model, and additionally, it showed how sensitive the stress fiber orientation in the experimental system is to the location where it is measured, i.e., the heterogeneity of the stress fiber orientation. Computed and experimental stress fiber orientations showed good quantitative agreement in most regions. A strong local alignment near the locations where boundary conditions were enforced was observed for both uniaxially and biaxially constrained tissues. Excepting these regions, in biaxially constrained tissues, no preferred orientation was found and the distribution was independent of location. The stress fiber orientation in uniaxially constrained tissues was more heterogeneous, and stress fibers mainly oriented in the constrained direction or along the free edge. These results indicate that the stress fiber orientation in these constrained microtissues is mainly determined by the local mechanical environment, as hypothesized in our model, and also that the model is a valid tool to predict stress fiber orientation in heterogeneously loaded tissues. © 2014 Springer-Verlag Berlin Heidelberg.