PURPOSE OF REVIEW: This review summarizes current and novel virologic reagents employed for the development and application of in-vitro assays that assess neutralizing activity of antibodies against HIV-1. Characteristics of several virologic approaches are placed in context with various cellular targets and assay read-outs intended to determine potency and breadth of neutralization in patient cohorts and clinical vaccine trials. RECENT FINDINGS: New molecular virologic reagents developed for in-vitro primary cell-based assays promise to facilitate rigorous and standardized assessment of anti-HIV-1-neutralizing antibody responses elicited by vaccine immunogens. SUMMARY: Comprehensive assessment of anti-HIV-1 antibody potency and breadth is essential for evaluating vaccine immunogens, the advancement of vaccine candidates into clinical trials, and ultimately the development of effective vaccine strategies. Env-pseudovirion and recombinant reporter cell line neutralization assays are important tools for rapid and standardized measurement of neutralizing antibody activity. However, recent studies indicate that reporter cell lines fail to detect neutralization activity of certain antibodies observed when analyzed in peripheral blood mononuclear cells and may yield results on neutralizing antibody breadth that are discordant with peripheral blood mononuclear cell assays. Importantly, it remains unknown whether current in-vitro assays may be predictive of a protective neutralizing antibody response elicited by vaccine immunogens. This situation underscores the significance of standardizing existing, complementary methods as well as developing new assay concepts that assess neutralization in primary cells. Thus, this chapter focuses on new virologic reagents that promise to facilitate reaching this goal. © 2009 Lippincott Williams & Wilkins, Inc.