This chapter involves an in-depth exploration of codon usage analysis, a crucial tool for understanding gene expression at the molecular level. It introduces several indices to evaluate codon usage preferences, diversity, and efficiency within genes. Key methods discussed include “base composition,” which analyses the relative proportions of DNA bases (A, T, C, G), and “relative synonymous codon usage” (RSCU), which measures the frequency of synonymous codons within genes and their impact on protein synthesis efficiency. Other indices, such as “frequency of optimal codons” (FOP), “codon bias index” (CBI), and “codon adaptation index” (CAI), offer insights into codon preferences, gene optimization, and translation efficiency. “Effective number of codons” (ENC) and “index of codon diversity (ICDI) assess codon usage diversity and natural variability in codon selection. These indices are fundamental in understanding how codon usage influences gene translation and evolution. The chapter also examines the evolution of codon usage in viruses, with a case study on influenza A virus, highlighting the relevance of these tools in virology. In conclusion, the chapter emphasizes the significance of codon usage analysis in both gene- and virus-related research and outlines potential directions for future exploration.