Recombinant miniature spidroin, RepCT, derived from the nursery web spider, Euprosthenops australis, was purified using protocols developed for ÄKTA pure chromatography system. To facilitate chromatographic purification, the miniature spidroin was fused to a solubility protein and histidine tag resulting in HisSolRepCT. After induced protein expression in Escherichia coli (E. coli), harvesting using centrifugation, cell lysis, and clarification, the clarified lysate was loaded onto a HiTrap™ IMAC HP column, captured, and eluted using step gradients with increasing concentrations of imidazole. Proteolytic on-column tag cleavage was simplified by the predefined affinity chromatography method in UNICORN™ v6.3 software. After desalting, the miniature spidroin, RepCT, was subjected to a polishing step consisting of anion exchange chromatography or gel filtration. This purification strategy resulted in a purity of RepCT exceeding 95% and a recovery of 85%. Identification of RepCT was confirmed by nanoscale LC-MS/MS and biological activity of the miniature spidroin was confirmed by the ability to assemble into macroscopic fibers.