Potassium channels display considerable functional diversity. Alternative pre-mRNA splicing represents one of the most powerful post-transcriptional mechanisms to create physiological diversity by generating multiple protein products from a single gene. Due to the modular nature of proteins, alternative splicing can profoundly modify potassium channel structure, function and regulation. Alternative pre-mRNA splicing is exploited by most genes but is particularly prevalent in single gene families as exemplified by the gene (KCNMA1), which encodes large conductance calcium- and voltage-gated potassium (BK) channel alpha-subunits. Importantly, alternative pre-mRNA splicing is kept under spatiotemporal control by circulating hormones and cellular activity, as well as being differentially modified during development and in different tissues. While the sequencing of numerous genomes has further demonstrated the importance of splicing in generating diversity from a limited genome size, a major challenge is to define splice variants that are expressed in tissues and their functional role. Here we describe strategies and protocols to experimentally define and isolate splice variant mRNA transcripts in multiple tissues and provide a platform to characterise the effect of splice variants on channel function and physiology.