DNA vaccines have become a reliable and major means to elicit immune responses in the past decade. We and others have attempted to obtain stronger, more long lasting, and optimized immune responses, subsequent to the pioneering works demonstrating the ability of plasmid DNA to raise specific immune responses. Advances in molecular biology and biotechnology allow the application of various adjuvants, immunologic agents that increase the antigenic response, in DNA vaccines. Adjuvants can be broadly separated into two classes based on their origin-genetic and conventional. Genetic adjuvants are expression vectors of cytokines or other molecules that can modulate immune responses when administered with a vaccine antigen. Conventional adjuvants are chemical compounds that enhance, prolong, or modulate antigen-specific immune responses. The use of an appropriate adjuvant is pivotal in optimizing the response to DNA vaccines. Moreover, DNA vaccines themselves possess their own adjuvant activity because of the presence of unmethylated CpG motifs in particular base contents. The route of inoculation is also a critical factor in determining the outcome of vaccination. It is well known that intramuscular injection preferentially induces Th1-type immunity, whereas particle bombardment by gene gun predominantly induces Th2-type response. This article focuses on providing the detailed procedure to construct genetic adjuvant plasmids and prepare DNA vaccines formulated with conventional adjuvants. We also offer a practical guide for the procedure of intramuscular DNA injection.