Alternative splicing of pre-mRNAs expands the coding abilities of genomes by generating distinct transcription variants from individual genes. It contributes to the marvelous complexity of the transcriptome in neurons. Given the differential expression of alternative splicing regulators and diversity in alternative splicing programs in neuronal subpopulations, it is urgent and necessary to develop methods to efficiently isolate diverse subgroups of neurons and analyze their transcriptomic diversity. Here, we describe a protocol to isolate RNA from specific neuronal types using a fluorescence-activated cell sorting (FACS)-based method to analyze alternative splicing events in a cell type–specific manner. The method is universally applicable to analyze alternative splicing in fluorescent protein-labeled neuronal types. It was optimized to preserve the transcription state and improve efficiency in cell suspension purification. With our protocol, fluorescent protein-labeled neurons could be efficiently purified. The transcription states suitable for gene expression and alternative splicing analysis could be well-preserved.