A systematic study of two-step CIEF analysis was completed to identify key components that could be optimized to enhance the performance of mAb analysis by CIEF. Resolution between mAb isoforms was increased by selecting a narrow detector aperture, utilizing chemical rather than pressure mobilization, and improving protein solubility by incorporating urea into the carrier ampholyte (CA) solutions. Loss of the extreme pI CAs and sample components by the bidirectional ITP inherent to IEF was avoided by setting the concentration of the phosphoric acid anolyte to 200 mM and sodium hydroxide catholyte to 300 mM and by adding sufficient amounts of an acidic (pI<3) and basic (10<pI) sacrificial ampholyte to the CIEF sample solution. Optimization of the concentrations of the sacrificial ampholytes, iminodiacetic acid and arginine to 1.7 and 40 mM, respectively, yielded a stable focused CA train that spanned the 4<pH<10 range without sacrificing either mAb isoform resolution or sample throughput. Intermediate precision studies performed on the CIEF method with three basic mAbs yielded 0.04 to 0.09% CVs for the estimated pI values and 0.6−3% CVs for isoform group percent composition, indicating that the two-step CIEF method developed meets the rigorous demands of therapeutic mAb analysis.