The recently released Linear Array human papillomavirus (HPV) genotyping test (Roche Diagnostics) provides a standardized method for simultaneous detection of up to 37 individual HPV types. This test offers a rapid approach for detecting and longitudinal monitoring of patients infected with high-risk (HR) HPV. However, it cannot rule out HPV52 infections in the presence of carcinogenic HPV genotypes 33, 35 and/or 58. As such, often only a non-definitive result of HPV52 presence can be reported. This study describes the development of a real-time PCR assay using an HPV52-specific hydrolysis probe in conjunction with the newly released Roche LightCycler 480 system. HPV52 was readily detected among DNA extracts from samples previously identified with possible HPV52 by the linear array test. Specificity was analyzed using a panel of DNA extracts previously identified as containing single/multiple HPV types, with or without HPV52. This is a rapid and simple assay, which could be used as a supplementary test to the Linear Array, to verify the presence or absence of HPV52 among samples testing positive for either HPV33, 35, and/or 58. Laboratories already using the Linear Array HPV genotyping test could adopt this method once internally validated.