Since 1996, A/goose/Guangdong/1/96-lineage highly pathogenic avian influenza (HPAI) H5 viruses have been causing flu outbreaks in poultry and wild birds. Occasionally, humans also fall victim to it, which results in high mortality. Nonetheless, HPAI virus research is often hindered, considering that it must be handled within biosafety level 3 laboratories. To address this issue, pseudoviruses are adopted as an alternative to wild-type viruses in some experiments of H5 HPAI studies. Pseudoviruses prove to be the ideal tools to study neutralizing antibodies against H5 HPAI viruses. This protocol describes the procedures and critical steps of H5 HPAI pseudovirus preparations and pseudovirus neutralization assays. Also, it discusses the troubleshooting, limitation, and modifications of these assays.