Certain host cell proteins (HCPs) in biotherapeutic drugs may be detrimental to drug product quality even when they are present at the subppm level. Therefore, an analytical method that can reliably quantify trace amounts of HCPs is desirable. This study demonstrates a novel strategy to quantify HCPs present at subppm levels with ProteoMiner enrichment coupled with limited digestion followed by targeted analysis with nano-liquid chromatography-parallel reaction monitoring. The method can achieve LLOQ values as low as 0.06 ppm, with an accuracy of 85%-111% of the theoretical value, and inter-run and intrarun precision within 12% and 25%, respectively. The approach was applied to the quantification of five high-risk HCPs in drug products. The results indicated that 2.5 ppm lysosomal acid lipase, 0.14 ppm liver carboxylesterase, 1.8 ppm palmitoyl-protein thioesterase 1, and 1 ppm cathepsin D affected the stability of drug products, whereas drug products could safely contain 1.5 ppm lipoprotein lipase, 0.1 ppm lysosomal acid lipase, or 0.3 ppm cathepsin D. In combination with lipase activity analysis, the accurate quantification of lipases/esterases in drug products enables better understanding and comparison of the enzymatic activity of polysorbate degradation from endogenous proteins.