Understanding the mechanobiological aspects of cardiac fibroblast activation is of utmost importance to elucidate the mechanisms underlying the onset and progression of cardiac disease. In this work, a protocol of hypoxia/reoxygenation (H/R) was applied to human cardiac fibroblasts to recapitulate, in vitro, the salient traits of cardiac fibrosis. Fibroblast activation and fibrotic ECM deposition were characterized via gene expression assays and immunofluorescence microscopy. Traction Force Microscopy (TFM) was also applied to measure cellular forces exerted by human cardiac fibroblasts (either normoxic or following H/R activation) on engineered hydrogel matrices. TFM is an imaging technique that quantifies spatially resolved interfacial forces through the analysis of the displacement field exerted by cells on an elastic substrate with known viscoelastic behavior. Traction forces were also evaluated using functional fluorescence traction sensors (modified vinculin protein featuring a FRET pair separated by an elastic segment) using spectral FRET microscopy. The combination of methodologies was successful in discriminating the effect of H/R on fibroblast activation, altered ECM deposition, and increased traction forces, hence representing a useful panel of techniques for the investigation of the mechanobiological aspects underlying cardiac disease.