Nucleoside phosphoramidites are derivatives of natural or synthetic nucleosides that are used to synthesize oligonucleotides, relatively short fragments of nucleic acid and their analogs. Oligonucleotides are widely used in biological research as effective tools to regulate gene expression and have also been developed as therapeutics. Therapeutic oligonucleotides are usually modified to enhance their stability and specificity and to minimize the side-effects. They include antisense oligonucleotide, small interference RNA (siRNA), ribozyme (RNA enzymes) and deoxyribozymes (DNA enzymes), CpG oligodeoxynucleotides (CpG ODN), transcription factor decoys, and aptamers. In various oligonucleotide synthesis methods, the phosphoramidite method is the most preferred one as it best meets the criteria for viable synthesis of oligonucleotides. The synthesis approach is suitable for the synthesis of long oligonucleotides with modified backbones, which are difficult to prepared by other methods. As long as a nucleoside analog contains at least one hydroxy group, it can be converted to the corresponding phosphamide by using appropriate protection strategies. The ability of phosphoramidites to undergo the phosphoramidite coupling reaction in the presence of an acidic catalyst makes phosphoramidites useful intermediates in oligonucleotide synthesis. The phosphoramidite strategy begins with the 3’-most nucleotide and proceeds through the cycles composed of fours steps (deprotection, coupling, capping, and stabilization).