This research article published by Engineering, Technology & Applied Science Research, Vol. 11, No. 3, 2021Sanger sequencing remains the cornerstone method for Deoxyribonucleic Acid (DNA) sequencing due to its high accuracy in targeting smaller genomic regions in a larger number of samples. The analysis of Sanger sequence DNA data requires powerful and intelligent software tools. Most of the preferred tools are proprietary licensed tools that offer a user-friendly interface and have many features, however, their affordability, especially to individual scientists or students, is limited. On the other hand, a few free and open-source licensed tools are available but have limited features. This study focuses on the usability testing of the developed Sanger Sequence Automatic Analysis Tool (SSAAT), a free and open-source web tool for Sanger sequence analysis. Usability tests were conducted with potential users and the results demonstrate that the participants were able to use the tool easily and accomplish the test tasks at the given time. Moreover, the participants were excited with the easy-to-use interface and agreed that most users could use the tool with no need for technical assistance. However, the participants also identified some issues that require more development effort.