Heparan sulfate (HS) and chondroitin sulfate (CS) glycosaminoglycans (GAG) are proteoglycan-associated polysaccharides with essential functions in animals. They have been studied extensively by genetic manipulation of biosynthetic enzymes, but chemical tools for probing GAG function are limited. HS and CS possess a conserved xylose residue that links the polysaccharide chain to a protein backbone. Here we report that, in zebrafish embryos, the peptide-proximal xylose residue can be metabolically replaced with a chain-terminating 4-azido-4-deoxyxylose (4-XylAz) residue by administration of UDP-4-azido-4-deoxyxylose (UDP-4-XylAz). UDP-4-XylAz disrupted both HS and CS biosynthesis and caused developmental abnormalities reminiscent of GAG biosynthesis and laminin mutants. The azide substituent of protein-bound 4-XylAz allowed for rapid visualization of the organismal sites of chain termination in vivo through bioorthogonal reaction with fluorescent cyclooctyne probes. UDP-4-XylAz therefore complements genetic tools for studies of GAG function in zebrafish embryogenesis.