Non-ionic surfactant polysorbates (PS), including PS-80 and PS-20, are commonly used in the formulation of biotherapeutic products for both preventing surface adsorption and acting as stabilizer against protein aggregation. Trace levels of residual host cell proteins (HCPs) with lipase or esterase enzymatic activity have been shown to degrade polysorbates in biologics formulation. The measurement and control of these low-abundance, high-risk HCPs for polysorbate degradation is an industry-wide challenge to achieve desired shelf-life of biopharmaceuticals in liquid formulation, especially for high-concentration formulation product development. Here, we reviewed the challenges, recent advances and future opportunities of analytical method development, risk assessment and control strategies for polysorbate degradation during formulation development with a focus on enzymatic degradation. Continued efforts to advance our understanding of polysorbate degradation in biologics formulation will help develop high-quality medicines for patients.