The objective of this study was to evaluate microflow imaging (MFI) as a sensitive tool to detect and quantify subvisible particle formation during freeze-thawing of an IgG(2) monoclonal antibody (mAb). Solutions of the protein formulated in 20 mM of histidine buffer (pH 5.5) were subjected to three freeze-thaw cycles and analyzed by MFI and size-exclusion chromatography (SEC). MFI showed increased particle numbers after each freeze-thaw cycle, whereas aggregates were not detected by SEC. Estimates of the total mass of particles formed revealed that monitoring of particle formation allows for the detection of protein aggregates comprising only hundredths of a percent of the total protein mass. Furthermore, differences in protein aggregation levels due to different formulations or different freeze-thawing protocols were resolved, even though protein aggregation could not be detected by SEC. To examine whether SEC and MFI-based estimations of total aggregate mass were in quantitative agreement, mAb was freeze-thawed in phosphate-buffered saline. This process created sufficient level of insoluble aggregates to be detected by SEC as a reduction in the monomer peak area in the chromatogram. There was good agreement between the loss of monomer detected by SEC and the total mass of subvisible particles detected by MFI.