Alternative splicing is a crucial post-transcriptional regulatory mechanism that generates multiple protein isoforms from a single gene, substantially increasing the coding capacity and proteome diversity of a genome. This process is particularly critical in regulating the activity of interleukin genes, where alternative splicing contributes to the functional diversity of these important immune system molecules and affects their production, function, and receptor interactions. While numerous studies have established the connection between aberrant alternative splicing and various diseases, including cancers and autoimmune disorders, the function and regulation of many splice variants remain poorly understood. Here, we describe a cost-effective and reliable method for analyzing alternative splicing patterns in interleukin genes using semi-quantitative RT-PCR and densitometry analysis. This method enables the simultaneous identification and quantification of multiple splice variants in a single PCR reaction, offering advantages over real-time RT-PCR approaches that require specific primer sets for each variant. This protocol involves RNA extraction from tissue culture cell lines or tissue samples, reverse transcription, RT-PCR, and subsequent analysis using freely available software for densitometry. We demonstrate the utility of this approach through two distinct examples with different alternative splicing patterns. While less sensitive than real-time RT-PCR or radioactive methods, this technique provides a robust, accessible, and widely accepted approach for investigating alternative splicing patterns in interleukin genes, contributing to our understanding of cytokine biology and its role in health and disease.