Aggregation and removal of detrimental substances from peroxide-bleached, fiber-free thermomechanical pulp water by dissolved air flotation (DAF) were studied for different chemicals. A focused beam reflectance measurement (FBRM) instrument was used to continuously assess the aggregation and removal of dissolved and colloidal substances (DCS) during batchwise DAF of process water. FBRM is particularly suitable for assessing the aggregation of DCS during the coagulation process prior to flotation. Differences between flocculants, as well as between different flocculant doses, can be observed. The FBRM results were in good agreement with turbidity measurements and with determination of wood resin and pectic acids by gas chromatography. General trends in the flotation process were also observed. However, monitoring the aggregates during flotation is more complicated due to the interference of air bubbles. To determine the overall flotation efficiency, a two-probe system with measurement points before and after actual flotation is proposed. It is essential to ensure good agitation at the measurement points, and if the destabilization of DCS produces sticky flocs, as in the case of cationic polyacrylamide, the position and the cleaning of the FBRM probe in the process pipe must be optimized to minimize the deposition of sticky material on the probe.