Injuries to tendons and ligaments are prevalent and result in a significant decrease in quality of patient life. Tissue-engineering strategies hold promise as alternatives to current treatments for these injuries, which often fail to fully restore proper joint biomechanics and produce significant donor site morbidity. Commonly, tissue engineering involves the use of a three-dimensional scaffold seeded with cells that can be directed to form tendon/ligament tissue. When determining the success of such approaches, the viability and proliferation of the cells in the construct, as well as extracellular matrix production and structure should be taken into account. Histology and histochemistry, microscopy, colorimetric assays, and real-time reverse transcriptase-polymerase chain reaction (RT-PCR) are techniques that are employed to assess these biological characteristics. This review provides an overview of each of these methods, including specific examples of how they have been used in evaluation of tissue-engineered tendon and ligament tissue. Basic physical principles underlying each method and advantages and disadvantages of the various techniques are summarized.