Surface modification of adenovirus vectors can improve tissue selective targeting, attenuate immunogenicity, and enable imaging of particle biodistribution; thus significantly improving therapeutic potential. Currently, surface engineering is constrained by a combination of factors including impact on viral fitness, limited access to functionality, or incomplete control over the site of modification. In order to alleviate this complication, our aim was to prelabel the virions metabolically with unnatural residues, which allow access to highly chemoselective reactions. Due to their small size, permissibility within biosynthetic pathways, and access to reactions with high specificity, azides and alkynes act as excellent bio-orthogonal handles. We utilized two different classes of unnatural residues, sugars and amino acids to metabolically label the capsid of adenovirus particles. The introduced substrates contain a chemical handle that can subsequently be modified with suitable probes. O-N-acetylglucosamine (GlcNAc) surrogate Ac4GalNAz affords specific incorporation of azide tag on the virus capsid while amino acids Azidohomoalanine and Homopropargylglycin make available a high concentration of modifiable substrates on the viral surface. Copper catalyzed bio-conjugation and Staudinger ligation reactions were used to decorate the particles with small molecules, peptides and even proteins. Incorporation of the unnatural substrates and the subsequent chemistries were characterized by western, fluorescence, enzymatic and mass spectrometry based assays. Analysis of viral infectivity by plaque forming assays showed substrate incorporations to have minimal effects on viral fitness. Targeted gene delivery capacities of the modified particles were analyzed by chemically attaching a targeting ligand on the virion surface. For this we initially utilized a folic acid based targeting system. After chemoselective attachment of this ligand onto virions containing GFP or Luciferase transgenes, the virions were targeted to breast cancer cell line, where we observed 20-fold higher levels of transgene expression compared to non-targeted particles. We also produced a new dual functional virus that contains 2 different bio-orthogonal tags and utilizing this system to probe applications into chemo and gene therapy based combination effects on tumor cell viability.