Abstract Summary Piwi-interacting RNAs (piRNAs) are a class of small non-coding RNAs which guide endonucleases to mRNAs of actively transcribed transposons in order to prevent their translation. The resulting mRNA fragments induce a positive feedback loop (the ‘ping-pong cycle’), which reinforces piRNA production and hence the transposon-silencing effect. PingPongPro is a command-line tool to scan small RNA-Seq data for signs of ping-pong cycle activity. It implements a novel algorithm that combines empirical probabilities in a multi-factor model to accurately identify transposons which are suppressed through the ping-pong cycle. Availability and implementation Source code, a user manual, and binaries for Microsoft Windows and Linux are available at https://github.com/suhrig/pingpongpro under the GPLv3 license. Supplementary information Supplementary data are available at Bioinformatics online.