Hep/HS disaccharide standards are essential disaccharide references for analyzing the disaccharide compositions of Hep/HS by enzymatic degradation combined with anion exchange HPLC. We use heparinases to selectively degrade various Hep/HS, combined with gel filtration and anion exchange HPLC, to prepare high-purity C4,5 unsaturated Hep/HS disaccharide standards with various sulfation patterns. These include eight common Hep/HS unsaturated disaccharides from digestion of Hep/HS by heparinases: I-A (ΔUA2S1→4GlcNAc6S), II-A (ΔUA1→4GlcNAc6S), III-A (ΔUA2S1→4GlcNAc), IV-A (ΔUA1→4GlcNAc), I-S (ΔUA2S1→4GlcNS,6S), II-S (ΔUA1→4GlcNS,6S), III-S (ΔUA2S1→4GlcNS) and IV-S (ΔUA1→4GlcNS); unsaturated disaccharides resulting from the deacetylation of Hep/HS during processing: I-H (ΔUA2S1→4GlcNS,6S), II-H (ΔUA1→4GlcNS,6S), III-H (ΔUA2S1→4GlcN) and IV-H (ΔUA1→4GlcN); and their 2AB-labeled derivatives.