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The potential of visible−near-infrared (Vis−NIR) spectroscopy to predict physico-chemical quality traits in 368 samples of bovine musculus longissimus thoracis et lumborum (LTL) was evaluated. A fibre-optic probe was applied on the exposed surface of the bovine carcass for the collection of spectra, including the neck and rump (1 h and 2 h post-mortem and after quartering, i.e., 24 h and 25 h post-mortem) and the boned-out LTL muscle (48 h and 49 h post-mortem). In parallel, reference analysis for physico-chemical parameters of beef quality including ultimate pH, colour (L, a*, b*), cook loss and drip loss was conducted using standard laboratory methods. Partial least-squares (PLS) regression models were used to correlate the spectral information with reference quality parameters of beef muscle. Different mathematical pre-treatments and their combinations were applied to improve the model accuracy, which was evaluated on the basis of the coefficient of determination of calibration (R²C) and cross-validation (R²CV) and root-mean-square error of calibration (RMSEC) and cross-validation (RMSECV). Reliable cross-validation models were achieved for ultimate pH (R²CV: 0.91 (quartering, 24 h) and R²CV: 0.96 (LTL muscle, 48 h)) and drip loss (R²CV: 0.82 (quartering, 24 h) and R²CV: 0.99 (LTL muscle, 48 h)) with lower RMSECV values. The results show the potential of Vis−NIR spectroscopy for online prediction of certain quality parameters of beef over different time periods.