The Spike (SARS-CoV-2) (B.1.351) Pseudotyped Lentivirus were produced with SARS-CoV-2 B.1.351 Variant Spike (Genbank Accession #QHD43416.1 with B.1.351 mutations (L18F, D80A, D215G, R246I, K417N, E484K, N501Y, D614G, A701V) as the envelope glycoproteins instead of the commonly used VSV-G. These pseudovirions contain the firefly luciferase gene driven by a CMV promoter, therefore, the spike-mediated cell entry can be measured via luciferase activity. The Spike (SARS-CoV-2) (B.1.351) pseudotyped lentivirus can be used to measure the activity of neutralizing antibody against SARS-CoV-2 B.1.351 variant in a Biosafety Level 2 facility.