Alternative splicing (AS) is a critical mechanism in gene regulation, generating transcript diversity and influencing protein structure and function. AS produces distinct splice isoforms across tissues, developmental stages, and conditions, playing a key role in biological processes and diseases. AS-related RNA-binding proteins (RBPs) regulate splicing events, affecting protein structure, function, and localization. Under physiological conditions, AS and RBPs contribute to tissue development and organ differentiation. Under pathological conditions, AS alterations are linked to diseases, particularly cancer, where abnormal splicing can lead to dysfunctional proteins. AS and RBPs are also associated with neurodegenerative and autoimmune diseases. This review summarizes recent advances in understanding AS and AS-related RBPs in tissue development and disease, highlighting their roles in gene expression complexity and precision medicine. AS is regulated by the spliceosome, a complex involving small nuclear RNAs and proteins. Splicing regulatory elements (SREs) in introns and exons regulate AS by binding to trans-acting factors. RBPs interact with RNA to form ribonucleoprotein complexes, influencing splicing, polyadenylation, RNA stability, and translation. RBPs are involved in various physiological and pathological processes, with defects in RBP function linked to diseases such as neurodegeneration, autoimmune disorders, and cancer. Analysis of AS in 8705 cancer patients revealed significant upregulation of alternative splicing events (ASEs) in pan-cancer cells. RBPs can induce exon inclusion or exclusion or alternative use of 5' or 3' splice sites by binding to pre-mRNA exons or flanking introns. Key components of signal transduction pathways also exhibit ASEs that regulate biological functions such as cell growth, development, differentiation, migration, and apoptosis. Recent advancements in AS-related RBPs include their role in regulating ASEs through distinct RNA sequences and structures, their involvement in normal developmental processes, their role in disease progression, and the use of advanced technologies to visualize and pinpoint ASEs. RBPs such as SRSFs and HNRNPs regulate AS by acting competitively in selecting splicing sites. AS-related RBPs are also involved in the regulation of epigenetic markers, such as histone modifications and DNA methylation, which influence exon usage and splicing site selection. The study of AS and RBPs has led to the identification of new therapeutic strategies, including the development of CRISPR-based RNA proximity proteomics (CBRPP) to identify proteins associated with endogenous RNA. High-throughput methods, such as transcriptomic data analysis, have improved our understanding of AS and RBPs, revealing their roles in various diseases. Future research may focus on the impact of DNA methylation and histone modifications on AS-RBPs, as well as the development of new technologies to study these interactions. The role of AS and RBPs in tissue development, particularly in the reproductive, neural, digestive, and immune systems, is also highlightedAlternative splicing (AS) is a critical mechanism in gene regulation, generating transcript diversity and influencing protein structure and function. AS produces distinct splice isoforms across tissues, developmental stages, and conditions, playing a key role in biological processes and diseases. AS-related RNA-binding proteins (RBPs) regulate splicing events, affecting protein structure, function, and localization. Under physiological conditions, AS and RBPs contribute to tissue development and organ differentiation. Under pathological conditions, AS alterations are linked to diseases, particularly cancer, where abnormal splicing can lead to dysfunctional proteins. AS and RBPs are also associated with neurodegenerative and autoimmune diseases. This review summarizes recent advances in understanding AS and AS-related RBPs in tissue development and disease, highlighting their roles in gene expression complexity and precision medicine. AS is regulated by the spliceosome, a complex involving small nuclear RNAs and proteins. Splicing regulatory elements (SREs) in introns and exons regulate AS by binding to trans-acting factors. RBPs interact with RNA to form ribonucleoprotein complexes, influencing splicing, polyadenylation, RNA stability, and translation. RBPs are involved in various physiological and pathological processes, with defects in RBP function linked to diseases such as neurodegeneration, autoimmune disorders, and cancer. Analysis of AS in 8705 cancer patients revealed significant upregulation of alternative splicing events (ASEs) in pan-cancer cells. RBPs can induce exon inclusion or exclusion or alternative use of 5' or 3' splice sites by binding to pre-mRNA exons or flanking introns. Key components of signal transduction pathways also exhibit ASEs that regulate biological functions such as cell growth, development, differentiation, migration, and apoptosis. Recent advancements in AS-related RBPs include their role in regulating ASEs through distinct RNA sequences and structures, their involvement in normal developmental processes, their role in disease progression, and the use of advanced technologies to visualize and pinpoint ASEs. RBPs such as SRSFs and HNRNPs regulate AS by acting competitively in selecting splicing sites. AS-related RBPs are also involved in the regulation of epigenetic markers, such as histone modifications and DNA methylation, which influence exon usage and splicing site selection. The study of AS and RBPs has led to the identification of new therapeutic strategies, including the development of CRISPR-based RNA proximity proteomics (CBRPP) to identify proteins associated with endogenous RNA. High-throughput methods, such as transcriptomic data analysis, have improved our understanding of AS and RBPs, revealing their roles in various diseases. Future research may focus on the impact of DNA methylation and histone modifications on AS-RBPs, as well as the development of new technologies to study these interactions. The role of AS and RBPs in tissue development, particularly in the reproductive, neural, digestive, and immune systems, is also highlighted