The majority of human genes undergo alternative splicing to produce multiple isoforms with distinct functions. The dysregulations of alternative splicing have been found to be closely associated with various human diseases; thus new approaches to modulate disease-associated splicing events will provide great therapeutic potentials. Here we report protocols for constructing novel artificial splicing factors that can be designed to specifically modulate alternative splicing of target genes. By following the method outlined in this protocol, it is possible to design and generate artificial splicing factors with diverse activities in regulating different types of alternative splicing. The artificial splicing factors can be used to change splicing of either minigenes or endogenous genes in cultured human cells, providing a new strategy to study the regulation of alternative splicing and function of alternatively spliced products.