Current techniques for monitoring glucose concentration during cell culture are invasively performed using an off-line measurement system. Even though <i>in situ</i> or <i>ex situ</i> analyte sensors have been tried for on-line measurement, they suffer from difficulties including the inability to keep them sterile, their limited lifetime, and their lack of stability. In this research an alternative optical noninvasive on-line monitoring system based on near-infrared absorbance spectroscopy was developed to measure glucose concentration of cell culture media in a rotary cell culture system. The system included an automatic sampling component controlled by a LabVIEW program and a high-throughput fiber coupling component connected to a Fourier transform near-infrared spectrometer. The effectiveness of the system was investigated via noninvasive on-line measurement of glucose absorbance spectra in the cell culture media during T-cell culture. Glucose absorbance spectra were collected in the spectral range of 2.0 to 2.5 μm. Partial least-squares regression was employed to build a successful multivariate calibration model. The standard error of prediction and mean percent error for glucose were 7.7 mg/dL and 1.0%, respectively. The successful results represent an important step in the development of a noninvasive, closed-loop, system for monitoring analytes in cell culture.

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