We present an R based pipeline, ArrayExpressHTS, for pre-processing, expression estimation and data quality assessment of high-throughput sequencing transcriptional profiling (RNA-seq) datasets. The pipeline starts from raw sequence files and produces standard Bioconductor R objects containing gene or transcript measurements for downstream analysis along with web reports for data quality assessment. It may be run locally on a users own computer or remotely on a distributed R-cloud farm at the European Bioinformatics Institute. It can be used to analyse users own datasets or public RNA-seq datasets from the ArrayExpress Archive.