The goal of this study is to evaluate the ability of a bimodal technique integrating time-resolved fluorescence spectroscopy (TRFS) and ultrasound backscatter microscopy (UBM) for nondestructive detection of changes in the biochemical, structural, and mechanical properties of self-assembled engineered articular cartilage constructs. The cartilage constructs were treated with three chemical agents (collagenase, chondroitinase-ABC, and ribose) to induce changes in biochemical content (collagen and glycosaminoglycan [GAG]) of matured constructs (4 weeks); and to subsequently alter the mechanical properties of the construct. The biochemical changes were evaluated using TRFS. The microstructure and the thickness of the engineered cartilage samples were characterized by UBM. The optical and ultrasound results were validated against those acquired via conventional techniques including collagen and GAG quantification and measurement of construct stiffness. Current results demonstrated that a set of optical parameters (e.g., average fluorescence lifetime and decay constants) showed significant correlation (p<0.05) with biochemical and mechanical data. The high-resolution ultrasound images provided complementary cross-section information of the cartilage samples morphology. Therefore, the technique was capable of nondestructively evaluating the composition of extracellular matrix and the microstructure of engineered tissue, demonstrating great potential as an alternative to traditional destructive assays.