Splicing is the most frequently altered biological process by mutations within gene regions. Information for splicing is recognized by several factors that bind pre-mRNA sequence and, through coordinated interaction, yield mature transcripts. Some in silico methods have been developed to predict if a mutation leads to aberrant splicing patterns. We previously created SpliceAid tool that is able to minimize false positive predictions because it adopts strictly experimental RNA target motifs bound by splicing proteins in humans. In order to improve prediction accuracy and better understand the splicing outcome, the tissue specificity of each splicing regulatory factor has to be taken into account. Here, we have developed SpliceAid 2 by adding the expression data related to the splicing factors extracted from the main proteomic and transcriptomic databases, true 5′ and 3′ splice sites, polypyrimidine tracts, and branch point sequences. The new version collects 2,220 target sites of 62 human splicing proteins and their expression data in 320 tissues per cell. SpliceAid 2 can be useful to foresee the splicing pattern alteration, to guide the identification of the molecular effect due to the mutations and to understand the tissue-specific alternative splicing. SpliceAid 2 is freely accessible at www.introni.it/spliceaid.html. Hum Mutat 33:81–85, 2012. © 2011 Wiley Periodicals, Inc.