Abstract Background Novel serological methods provide alternative options for sero-diagnosis, sero-epidemiology and for determining evidence of naturally acquired or vaccine induced immunity. Micro-neutralization tests are currently the gold standard for serological studies of highly pathogenic avian influenza in mammalian species but require handling live virus in a biosafety level (BSL) 3 environment. We previously reported the use of H5 pseudotyped lentiviral particles (H5pp) as an alternative to micro-neutralization tests in a BSL-2 setting (Nefkens et al., 2007). Objective To optimize and evaluate this newly developed H5pp assay on relevant clinical specimens. Study design We optimise and evaluate the performance of the H5pp assay using well-characterized sera from humans with confirmed H5N1 disease or controls. Results The H5pp assay is a reliable serological method for the detection and quantification of neutralizing antibody to H5-viruses. Conclusion H5pp provide a reliable and safe alternative for sero-diagnosis and sero-epidemiology of H5N1 infections in a BSL-2 setting.