Purpose: During biotherapeutics manufacturing, process-related impurities and other trace contaminants can be present along with recombinant biotherapeutic products. Among them, host cell proteins (HCPs) are a major type of protein impurity derived from the host organism. The detection and quantification of HCPs is an area of particular concern, as these contaminants can elicit an adverse response in patients. The high complexity and the wide dynamic range of protein concentrations in the multiple purification stages of biotherapeutic production poses challenges for the traditional data dependent workflows for HCP quantification. Therefore, targeted quantitation approaches with high sensitivity have attracted more interest as a quality control. Herein, a comprehensive solution for targeted HCP quantitation utilizing various LC-MS platforms is presented, to determine an optimal analytical methodology. Enhanced quantitative performance is achieved by use of a new triple quadrupole LCMS system with E-Len and D-Jet ion focusing elements to drive improved S/N, low end LDR and CVs. Improved LLOQ is observed in the range of 4X compared to standard triple quadrupole systems. Methods: The NIST monoclonal antibody (NISTmAb) standard and additional protein standards are spiked into the CHO cell culture media sample. The protein standards cover 5 orders of magnitude of abundances and serve as model proteins for HCP quantitation workflow development. The sample are denatured, reduced, alkylated, digested and subjected into LC-MS/MS analysis. Multiple LC-MS platforms, including Triple-Quad/QTRAP and Q-TOF high resolution mass spectrometers, are utilized for data acquisition. MRM, data-independent acquisition and high-resolution MRM acquisition modes are evaluated to achieve the ideal quantitation performance. Results: The protein spiked-in samples are digested and subjected to peptide mapping experiments using data dependent workflow by Q-TOF MS. The data was searched against CHO proteome database and blastp processing for signature peptide selection. The selection criteria are based on signal intensity, sequence uniqueness, and ease to be modified. The acquisition methods to quantify these signature peptides were developed by either monitoring the abundant fragment ions or using data-independent workflow. To develop the targeted acquisition methods, the most abundant fragment ions of the target HCP peptides are monitored. The compound parameters for each MRM transition are optimized by injection/infusion of digested protein standards. To develop data-independent acquisition method, the variable window SWATH® acquisition is used for optimal analyte coverage and fast cycle time for quantitation. The window widths are determined based on the MS ion intensity distribution. The quantitation performance of multiple LC-MS/MS workflows are compared by reviewing the LLOQs, dynamic ranges, precision, accuracy, linearity and reproducibility. Inter-sample dynamic ranges are evaluated by analyzing each individual protein with calibration curve generation with a serial dilution sample set. For intra-sample dynamic range, proteins with different spiked-in abundances are across-compared to evaluate the linearity. For MRM analysis, a sensitivity comparison is performed between a standard and a modified triple quadrupole system. Multiple hardware improvements are applied on the modified triple quadrupole system for enhanced sensitivity. By allowing significantly larger number of target precursor ions to transit the system, the modified triple quadrupole system shows significant improvement in sensitivity when quantifying HCP peptides in biological matrices. Conclusion: A targeted HCP quantitation workflow utilizing versatile LC-MS methodologies including a novel triple quadrupole system is reported.