A class of mutations, namely splice site mutations, disrupt RNA processing, leading to exon skipping, intron retention and altered isoform expression, which safeguards genomic stability and cellular homeostasis. TP53, a critical tumor suppressor gene, encodes for 9 mRNA transcripts and 12 protein isoforms. However, the precise molecular and clinical consequences of TP53 splice mutations remains poorly understood. We performed an in-silico analysis of 25,058 TP53 mutations from TCGA, MSK-MetTropism and TMB–Immunotherapy datasets (via cBioPortal) and germline variants from IARC and gnomAD. We compared mutation frequencies, nucleotide spectra, association of these splice mutations on TP53 mRNA, protein expression and copy number, expression of p53 target genes, pathway enrichment, tumor mutation burden and fraction of genome altered. Splice mutations were reported at donor sites X32, X125, X224, X261 and X331 and acceptor sites X33, X126, X187, X225, X307 and X332, with significant differences in their frequencies between somatic and germline cohorts. Functional annotation revealed two outcomes: (i) reduced TP53 mRNA with global downregulation of p53 targets mainly with donor sites splice mutations and (ii) preserved transcript levels with mixed downstream regulation, which comprised of acceptor site splice mutations. Specific splice mutations, notably X125 and X331, were linked to higher tumour mutation burden, increased genomic instability, than the flanking acceptor site mutations. To investigate functional consequences, we generated CRISPR-engineered lung cancer cell clones carrying the X125 splice mutation in varying gene dosages (25–50%). Basal analyses revealed reduced p53 protein abundance, consistent with a loss-of-function phenotype. Upon Nutlin-3A treatment, results demonstrated, canonical p53 targets including MDM2 and p21 were significantly downregulated at both mRNA and protein levels, indicating functional inactivation of the p53 pathway. Furthermore, we found that despite overall p53 protein being lower, there was a change in the amount of individual isoforms across the mutants. Collectively, this data suggests, X125 acts as a loss of function mutation, impairing p53 transcriptional activity while stabilising selective isoforms that contribute to tumour heterogeneity and altered stress responses. We are currently in the process of identifying the functional consequences of the presence of the X125 splice mutations. By integrating large-scale genomic analyses with isogenic functional models, we aim to elucidate how TP53 splice mutations drive tumorigenesis, and whether this is through genomic instability and p53 expression. This research in the future has the potential to pave the way for isoform-specific therapeutic strategies.