Human alpha-defensins are small, Cys-rich, cationic proteins expressed predominantly in neutrophils and intestinal epithelia. They play important roles in innate and adaptive immunity against infection. Progress in studying these molecules can be accelerated by access to large quantities of high-quality materials, which have been obtained mainly from natural sources. Here, we report total synthesis of human alpha-defensins 4, 5, and 6, also known as HNP4, HD5, and HD6, using the optimized N,N-diisopropylethylamine (DIEA) in situ neutralization/2-(1 H-benzotriazolyl)-1,1,3,3-tetramethyluroniumhexafluorophosphate (HBTU) activation protocol for solid-phase Boc chemistry. Oxidative folding/disulfide formation was achieved directly using crude peptides, resulting in an overall synthetic yield of 10-16% with high purity. Antimicrobial activity assays were performed with Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 29213, using colony-counting methods, and the results demonstrated differential activity against these strains. Our report describes a highly efficient synthetic approach that enables thorough structural and functional studies of these three important immunologic molecules.