The generation of traction forces by neutrophils regulates many crucial effector functions responsible for host defense, such as attachment, spreading, migration, phagocytosis, and NETosis. The activation state of the cell is a strong determinant of the functional efficacy of the neutrophil; however, the effect of activation on traction force production has not yet been determined experimentally. Previously, the mapping of cellular-generated forces produced by human neutrophils via a Traction Force Microscopy (TFM) method has required a three-dimensional imaging modality to capture out-of-plane forces, such as confocal or multiphoton techniques. A method newly developed in our laboratories can capture out-of-plane forces using only a two-dimensional imaging modality. This novel technique—combined with a topology-based single particle tracking algorithm and finite element method calculations—can construct high spatial frequency three-dimensional traction fields, allowing for traction forces in-plane and out-of-plane to the substrate to now be differentially visualized and quantified with a standard epifluorescence microscope. Here we apply this technology to determine the effect of neutrophil activation on force generation. Sepsis is a systemic inflammatory response that causes dysregulated neutrophil activation in vivo. We found that neutrophils from septic patients produced greater total forces than neutrophils from healthy donors and that the majority of this dysregulation occurred in-plane to the substrate. Ex vivo activation of neutrophils from healthy donors showed differential consequences depending on activation stimuli with mechanosensitive force decreases observed in some cases. These findings demonstrate the feasibility of epifluorescence-based microscopy in mapping traction forces to ask biologically significant questions regarding neutrophil function.