*Prost!* (PRocessing Of Short Transcripts) can analyze smallRNA sequencing data generated on any sequencing platform. *Prost!* does not rely on existing annotation to filter sequencing reads but instead starts by aligning all the reads on a user-provided genomic reference, allowing the study of miRNAs in any species. Additionally, any number of samples can be studied together in a single *Prost!* run, allowing an accurate analysis of an entire dataset. After grouping the processed reads by genomic location, *Prost!* then annotates them using a user-defined annotation database (public or personal annotation database). Genomic alignment, grouping, and then annotation enable the study of potentially novel miRNAs, as well as permitting the retention of all the isomiRs that a miRNA may display. Finally *Prost!* contains additional features such as grouping by seed sequence for a more functional approach of the dataset, provides automatic discovery of potential mirror-miRNAs, and analyzes the frequency of various types of post-transcriptional modifications at each genomic location. Each step of the *Prost!* analysis is provided in an Excel output file so that the user have access to all information for deeper analysis of specific cases.