The distortions in the high-order structure of therapeutic monoclonal antibodies (mAbs) under different environmental conditions acutely affect mAb stability, resulting in altered safety, efficacy, and shelf-life profiles. The overall stability of mAbs depends on many factors, and it re-quires complementary techniques for an in-depth analysis. The stability of mAbs can be character-ized by differential centrifugal sedimentation (DCS), differential scanning calorimetry (DSC), differential scanning fluorimetry (DSF), and size exclusion chromatography (SEC) techniques. In this report, temperature-ramped dynamic light scattering (DLS), and circular dichroism (CD) spectroscopy were employed as complementary tools to show how temperature and pH affect the aggregation of a model mAb, trastuzumab, in solution. The results showed that the aggregation onset temperature of trastuzumab defined by DLS was 75 °C, which decreases the amount of β-sheets and causes a slight increase in helix structures. Moreover, the melting temperature of trastuzumab was determined to be between 80–83 °C by temperature-ramped CD spectrophotometry, which is in line with the Tm of trastuzumab’s Fab region tested with DSC. Thus, unfolding and aggregation of trastuzumab start simultaneously at 75 °C, and unfolding triggers the aggregation. The tempera-ture-ramped CD and DLS methods are robust tools to determine the thermal behavior of biosimilars in various solution conditions. Their complementary usage provides solid scientific background for regulatory applications and a better understanding of mAb instability and its relationship with structural changes.