Pyrosequencing(®) is a widely used technology to detect gene mutations in a molecular research or diagnostics laboratory. Compared to Sanger sequencing, it is inherently more quantitative with a superior limit of detection, although it has a shorter read length and has difficulty with homopolymeric sequences.Results of Pyrosequencing experiments are typically presented as traces with sequential peaks, called Pyrograms(®). For the majority of clinical diagnostic cases, Pyrograms are straightforward to read. However, there are occasionally complex results that are uninterpretable or difficult to interpret. In this chapter, we demonstrate a computer software, named Pyromaker that has been developed to help with the analysis of Pyrograms. Pyromaker is a freely and publically available software program to assist in the recognition of patterns of mutations, interpretation of difficult or ambiguous testing results and design of an optimal strategy to detect potential mutations by generating simulated Pyrograms. In addition to help diagnostic activities, Pyromaker can also be used as a virtual and user-friendly educational tool to teach newcomers the fundamental mechanism of Pyrosequencing, and correct interpretation of actual Pyrosequencing data.