Abstract Motivation: Cancer researchers seeking immunotherapy targets in cancer cells need tools to locate highly expressed proteins unique to cancer cells. Missense mutation and frameshift location reporter (MMuFLR), a Galaxy-based workflow, analyzes next-generation sequencing paired read RNA-seq output to reliably identify small frameshift mutations and missense mutations in highly expressed protein-coding genes. MMuFLR ignores known SNPs, low quality reads and poly-A/T sequences. For each frameshift and missense mutation identified, MMuFLR provides the location and sequence of the amino acid substitutions in the novel protein candidates for direct input into epitope evaluation tools. Availability: http://toolshed.g2.bx.psu.edu/ Contact: rath0096@umn.edu or johns198@umn.edu Supplementary information: Supplementary data are available at Bioinformatics online.