CIEF with whole-column imaging detection (WCID) can be a useful tool for the characterization and identification of human papillomavirus (HPV). This article is the initial report of the determination of the pI of HPV by CIEF-WCID method. In this study, components of the assay selected for optimization were ampholytes, additives, methylcellulose concentration, HPV concentration, salt concentration, and focusing time. Then the optimization CIEF-WCID method was validated for HPV 16L1 and HPV 18L1. As a result, a precise method to analyze the pI values of HPVs was achieved with RSD < 1.0%. The HPV peak pattern was reproducible. CIEF-WCID had great potential for HPV quality control, as WCID eliminated the mobilization step required by the conventional single-point detection. In the example, the five HPVs displayed pI values of 8.43 +/- 0.06 (n = 10; HPV 6L1), 8.70 +/- 0.04 (n = 10; HPV 11L1), 7.94 +/- 0.05 (n = 18; HPV 16L1), 7.57 +/- 0.04 (n = 18; HPV 18L1), and 8.45 +/- 0.05 (n = 10; HPV 58L1). This CIEF-WCID platform could be a powerful analytical tool for characterization, process development support, release testing, and stability study in pharmaceutical industry.