Analytical　screening　and　validation　systems　based　on　a　combination　of　cell　membrane　chromatography　and　two-dimensional　chromatography-tandem　mass　spectrometry　are　incapable　of　providing　prepared　samples　containing　the　active　ingredients　found　in　traditional　Chinese　medicine;　therefore,　these　samples　cannot　be　directly　used　in　subsequent　studies.　In　this　study,　a　semi-preparative　cell　membrane　chromatography　column　was　developed　using　a　hydrogel-modified　carrier　and　human　umbilical　vein　endothelial　cells　to　optimize　prepared　conditions,　such　as　hydrogel　polymerization,　cell　fragmentation,　and　cell　membrane　volume.　This　increased　the　binding　ratio　of　membrane　protein　and　carrier　to　15.79 mg/g.　The　column　was　systematically　evaluated　using　multitarget　tyrosine　kinase　inhibitors　that　displayed　good　specificity　and　reproducibility.　Subsequently,　using　the　column　coupled　with　a　semi-preparative　high-performance　liquid　chromatography-offline-high-performance　liquid　chromatography-mass　spectrometry　system,　15　active　ingredients　were　screened　and　purified　from　Indigo　naturalis,　and　five　main　components　were　identified:　l-lysine,　oxyresveratrol,　tryptanthrin,　isorhamnetin,　and　indirubin.　Furthermore,　the　pharmacological　effects　of　the　ingredients　were　confirmed　using　cell　proliferation　and　apoptosis　assays.　Results　revealed　potent　proliferation-inhibiting　and　apoptosis-promoting　abilities　on　human　chronic　myelogenous　leukemic　cells　and　human　promyelocytic　leukemic　cells　(p 　©　2023　Wiley-VCH　GmbH.