Near-infrared (NIR) spectroscopic analysis for suspensions often requires a pretreatment by dilution or filtering of suspended solids to maintain the sensitivity of the measurements. An online ultrasound-assisted spectroscopy (UAS) unit enabling pretreatment-free and noncontact analysis for bioprocessing is proposed and evaluated with a model suspension containing 3-μm-diameter polystyrene latex particles (PSLs) with the density of 5.1 × 108 counts/ml and Chinese hamster ovary (CHO) cell culture whose cell density was 3.2 × 107 cells/ml. The online UAS uses acoustic radiation force generated by ultrasonic standing waves. Suspended matter such as the PSLs and CHO cells can be localized at nodal planes in the suspensions by the acoustic radiation force. Hence, in the case of the online UAS, incoming light can pass through the suspensions more easily than that in the case of a conventional spectroscopy. Its effectiveness was evaluated by the predictive capability of a calibration model for glucose concentrations in the model suspensions and the CHO cell cultures. The calibration models were constructed by use of a partial least-squares regression (PLS-R) in the range of 4900-4200 cm-1 region after the pretreatment of second-order Savitzky-Golay filter. The calibration model built from the NIR spectra acquired with the online UAS could predict the glucose concentration in the CHO cell cultures with a measurement error of 0.6%. It was validated that the glucose concentrations in the flowing model suspension were able to be monitored by the online UAS with a measurement error of 8%. The newly developed online UAS for cell culture monitoring allows us to promote a wider use of NIR spectroscopy. For example, in the applications to the biopharmaceutical and cell-therapy industries, the online UAS enables simpler and easier monitoring of cell cultures because cleaning and sterilization of monitoring tools after cell culturing can be eliminated.