Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) is an accurate and fast method to measure gene expression. Reproducibility of the analyses is the main limitation of RT-qPCR experiments. Galaxy is an open, web-based, genomic workbench for a reproducible, transparent, and accessible science. Our aim was developing a new Galaxy tool for the analysis of RT-qPCR expression data. Our tool was developed using Galaxy workbench version 19.01 and functions implemented in several R packages. We developed PIPE-T, a new Galaxy tool implementing a workflow, which offers several options for parsing, filtering, normalizing, imputing, and analyzing RT-qPCR data. PIPE-T requires two input files and returns seven output files. We tested the ability of PIPE-T to analyze RT-qPCR data on two example datasets available in the gene expression omnibus repository. In both cases, our tool successfully completed execution returning expected results. PIPE-T can be easily installed from the Galaxy main tool shed or from Docker. Source code, step-by-step instructions, and example files are available on GitHub to assist new users to install, execute, and test PIPE-T. PIPE-T is a new tool suitable for the reproducible, transparent, and accessible analysis of RT-qPCR expression data.