The present disclosure relates to an in-line variable pathlength spectrophotometer. Currently, at-line spectrophotometers are used to measure the concentration of substances in a liquid during manufacturing of various formulations. Since manufacturing needs to be paused for at-line measurements, at-line spectrophotometers create an undesirable overhead. An in-line spectrophotometer is attached to the bioreactor and measures the concentration of substances in the liquid contained in the bioreactor in real-time, without any need to pause manufacturing. Moreover, the pathlength of the spectrophotometer, namely the distance light travels through the measured liquid, may be adjustable. Hence, measurements may be taken multiple times with varied pathlength so that accuracy of measurements may be enhanced. In one embodiment, the spectrophotometer may rotate about a central axis of the bioreactor to minimize disturbance to the homogeneity of the liquid. In another embodiment, the spectrophotometer may be attached to a port on an inner wall of the bioreactor.