Acute Myeloid Leukemia (AML) is a hematological malignancy in which there is an over-proliferation of immature myeloid cells. AML is commonly treated with the Bcl-2 inhibitor Venetoclax, but around 30% of patients fail to respond and most eventually develop resistance. Cells can develop resistance to Venetoclax through increased mitophagy, the digestion of damaged mitochondria, which is regulated by alternative splicing of intron 6 in the PINK1 gene. We hypothesized that the inhibition of PINK1 splicing using antisense oligonucleotides (ASOs) would decrease mitophagy and enhance cancer cell sensitivity to Venetoclax. K-562 AML cancer cell lines were transfected with an ASO targeting the 5’ junction between exon 6 and intron 6. Visualization of splicing through PCR and agarose gel electrophoresis indicated significant reduction of PINK1 splicing. qPCR confirmed reduced levels of mature mRNA levels upon 5’ASO transfection while pre-mRNA levels remained unaffected, indicating regulation through non-sense mediated mRNA decay without affecting transcription levels. Significant reduction of PINK1 splicing was also maintained upon treatment with Venetoclax. Flow cytometry analysis of apoptosis through staining with Annexin V and 7-AAD confirmed enhanced cell death among cells treated with both Venetoclax and 5’ASO compared to cells treated only with Venetoclax. These data support our hypothesis that inhibition of mitophagy through PINK1 5’ASO transfection enhances cell sensitivity to Venetoclax, thereby presenting a possible targeted therapeutic advancement.