Alternative splicing significantly contributes to gene expression heterogeneity and disease progression, yet analyzing its dynamics in short genetic regions such as microexons remains challenging. Here we identified notable variations in ribosomal protein S24 (RPS24) splicing patterns across breast cancer subtypes and investigated this novel regulatory mechanism. To overcome the complexity of analyzing three consecutive microexons (3 bp, 18 bp and 22 bp), we developed a specialized approach combining splice junction read analysis with fragment analysis for accurate isoform quantification. We observed distinct isoform compositions across breast cancer cell lines. The 3-bp exon-containing isoform (ex4:3 bp) of RPS24 showed significantly higher expression in estrogen receptor-positive (ER⁺) cells, demonstrating the strongest association with estrogen receptor signaling among all analyzed genes. This isoform functions as a molecular sensor for therapeutic interventions, being consistently upregulated following mTOR or CDK4/6 inhibition but consistently reduced across diverse drug-resistant cell lines, regardless of resistance mechanism. Through systematic RNA-binding protein screening and crosslinking immunoprecipitation followed by high-throughput sequencing analysis, we identified PTBP1 as a critical upstream regulator mediating microexon skipping. Analysis of multiple patient cohorts demonstrated that decreased ex4:3 bp expression strongly correlated with poor epithelial differentiation and metastatic progression specifically in ER⁺ breast cancer. Our findings suggest that RPS24 alternative splicing is associated with a multilayered regulatory network integrating ER signaling, cell cycle and PTBP1-mediated splicing. The ex4:3 bp isoform serves as a potential biomarker for drug resistance and treatment response in ER⁺ breast cancer.