An optical fiber biosensor to detect calpastatin has been investigated as a preliminary step in developing tenderness detection instrumentation. Longissimus dorsi samples were taken from beef carcasses (n = 21) at 0, 24, 36 and 48 h postmortem. Muscle homogenates were assayed for calpastatin activity using traditional methods and an optical fiber biosensor. Warner-Bratzler shear force was also performed on a steak from each carcass at 14 d postmortem. Results demonstrated that the measurements with highest correlation between traditional calpastatin assays and optical biosensor readings were taken at 48 h postmortem (r = 0.597, P ≤ 0.01), suggesting that this is the best time for use of this biosensor in an on-line grading system. This research further advances the development of a calpastatin biosensor and would be useful in laboratory determination of the presence of biologically active calpastatin concentrations.