Transcriptome data is a cost-effective approach to obtaining the coding regions of the genome. Recent advances in sequencing technologies have led to a rapid expansion in newly acquired and publicly available transcriptome data. At the same time, advances in bioinformatics have led to computationally efficient approaches to cleaning and assembling transcriptome data. However, de novo assembly methods often require some fluency in programming if labs wish to perform them in-house. Here we present PAAnDO (Pipeline for the Assembly and Analysis of De Novo Transcriptomes), a user-friendly package enabling the streamlined assembly and differential gene expression analysis of de novo transcriptomes from short-read RNA sequence data for non-model organisms. Requiring no programming knowledge, PAAnDO utilizes the newest bioinformatic approaches to maximize the efficiency and accessibility of de novo transcriptome assembly for non-programmers.