Modern molecular biology approaches often result in the accumulation of abundant biological sequence data. Ideally, the function of individual proteins predicted using such data would be determined experimentally. However, if a gene of interest has no predictable function or if the amount of data is too large to experimentally assess individual genes, bioinformatics techniques may provide additional information to allow the inference of function. This chapter proposes a pipeline of freely available Web-based tools to analyze protein-coding DNA sequences of unknown function. Accumulated information obtained during each step of the pipeline is used to build a testable hypothesis of function. The basis and use of sequence similarity methods of homologue detection are described, with emphasis on BLAST and PSI-BLAST. Annotation of gene function through protein domain detection using SMART and Pfam, and the potential for comparison to whole genome data are discussed.