Although many splice sites have been identified by computational and in vitro splicing techniques, a comprehensive analysis tool for predicting the cryptic 5’ splice site activation in gene mutation was not available. iSplice is an online tool combining gapped-dinucleotide pattern probability with logarithmic odds (GO algorithm) to assess the likelihood of the activation of a cryptic 5’ splice site, which competes with its paired authentic 5’ splice site. This study showed that iSplice has better prediction rate than four other scoring methods, namely information contents (Ri), free energy (Δ
G), maximum entropy model (MaxEnt), and maximum dependence decomposition model (MDD). iSplice was a web-based tool that could calculate the possibility of various splicing events in designed mutagenesis experiments.