The Single Molecule Profiler (SMP) software automatically generates a database from the single molecule localization files that have been organized in a plate layout format, as illustrated by the tree folder hierarchy. ASCII metadata text files (i.e. DetectionsFile.txt, TracksFile.txt, ClusterFile.txt, etc.) must contain the same number of header lines, identical column separators and descriptors for all wells and all positions, all of which can be specified by the user. A single metadata text file is used as a model for the data format and is parsed to generate descriptors for each column in the file. The user may select which descriptors, as well as the maximum number of objects (detections, tracks or clusters...), to be included in the database to reduce the final database size. The Single Molecule Profiler automatically generates the all files (red rectangle) necessary for importing into Cell Profiler Analyst (http://cellprofiler.org/cp-analyst/).