The pseudo-neutralization assay (PsV) described in the current report allows for the creation of HPV18 pseudovirions in order to evaluate whether the antibody responses elicited following vaccination with Gardasil(®) are sufficient to neutralize the activity of these pseudovirions in vitro. The PsV assay evaluates a broader antibody response than the HPV competitive Lumniex Immunoassay (cLIA), which monitors the presence of a single neutralizing epitope. We employed two different approaches to the HPV18-PsV assay: one using standard dilutions of heat inactivated serum from vaccinated subjects, as is typically reported in the literature, and the other using heat inactivated serum from which IgG antibodies were purified and quantitated as an attempt to reduce assay background and achieve a level of quantitation greater than that afforded through simple dilution. Here, we show that after 48 mo, Gardasil(®) vaccinated subjects from three groups defined by HPV 18 cLIA titer have detectable HPV18 neutralizing antibodies as measured by either approach in the HPV18-PsV assay. These data support the observed sustained HPV18 protection against persistent infection and disease with the absence of breakthrough cases in Gardasil(®) vaccinees and suggests that neutralizing antibodies are present although they may no longer be detectable by cLIA.