Covering the ends of long bones, articular cartilage provides a smooth, lubricated surface to absorb impact and distribute loads during movement so that underlying bone is protected. This function is facilitated by a complex and well-organized extracellular matrix (ECM). Being the only cell type in articular cartilage, chondrocytes are responsible for maintaining the homeostasis of the cartilage ECM; as such, the viability of chondrocytes is a critical parameter to reflect the quality of the cartilage. Most prevalent cell viability assays rely on dye staining and thereby cannot be performed for longitudinal monitoring or in-vivo assessment. Here we demonstrate that two-photon autofluorescence (TPAF) microscopy distinguishes live cells from dead cells in intact ex-vivo cartilage tissues, which provides a non-invasive method to assess cell viability. In our study, the endogenous fluorophores such as nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) and flavin adenine dinucleotide (FAD) were used to image chondrocytes in cartilages on rat tibia condyles immediately after harvesting. Second harmonic generation (SHG) imaging was also performed to examine the integrity of the extracellular and pericellular matrix. On the same tissue, the cell viability assay with Calcein-AM and Ethidium homodimer-1 (EthD-1) labeling was used as a gold standard to identify live or dead cells. We found that live cells presented stronger NAD(P)H fluorescence than dead cells in general and were readily identified if pseudo colors were used for showing two-channel images. Owing to its non-destructive nature, this method holds the potential value in assessing cell viability of engineered or living tissues without dye labeling.