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Lentiviral vectors (LVs) are powerful tools in gene therapy that have recently witnessed an increasing demand in both research and clinical applications. Current LVs purification represents the main bottle neck in their application as several methods are employed which are time consuming, cumbersome and yield low recoveries. The aim of this project was to develop a one-step method to specifically and efficiently purify LVs, with high vector yields and reduced levels of impurities, using the biotin-streptavidin system. Herein, packaging 293T cells were genetically engineered with biotin mimicking synthetic peptides and different cell membrane anchoring strategies for optimal streptavidin binding were tested. We have identified a flanked disulphide-constrained peptide, termed Ctag (ECHPQGPPCIEGRK), displayed on a CD8α stalk to be the most promising. LVs were modified with Ctag by its random incorporation onto viral surfaces during budding, without viral protein engineering or hindrance on infectivity. The expression of Ctag on LVs allowed complete capture of infectious particles by streptavidin magnetic beads. As Ctag binds streptavidin in the nanomolar range, we hypothesised that gentle elution from streptavidin matrix should occur by biotin’s competitive binding. Accordingly, addition of micromolar concentrations of biotin to captured LVs resulted in an overall yield of ≥60%. Analysis of eluted LVs revealed high purity levels, with a ≤3-log and 2-log reduction of DNA contamination and host cell proteins, respectively. This one-step purification was also tested for scalable vector processing using streptavidin monolith affinity chromatography and preliminary results were encouraging with 20% overall yield. In conclusion, we developed a single-step affinity chromatography which allows specific purification and concentration of infectious vectors modified with a biotin mimic. Based on intended usage, efficient LV purification can be achieved using both magnetic beads and column chromatography. This method will be of valuable use for both research and clinical applications of LVs.