We report on the development of the high-throughput screening (HTS) Navigator software to analyze and visualize the results of HTS of chemical libraries. The HTS Navigator processes output files from different plate readers formats, computes the overall HTS matrix, automatically detects hits and has different types of baseline navigation and correction features. The software incorporates advanced cheminformatics capabilities such as chemical structure storage and visualization, fast similarity search and chemical neighborhood analysis for retrieved hits. The software is freely available for academic laboratories.