Epidermal keratinocytes, the major cell type of the epidermis, undergo a well-orchestrated balance between proliferation and differentiation, which is essential for proper skin function. While the related control processes have been studied at the protein-coding gene level, increasing evidence highlights the importance of long noncoding RNAs (lncRNAs) in regulating gene expression and cell fate decisions. LncRNAs constitute a large proportion of the human transcriptome; however, the biological functions of most of them remain poorly characterized. Emerging studies demonstrate that lncRNAs play critical roles in cellular identity, differentiation, and tissue homeostasis. In the epidermis, several lncRNAs have been shown to regulate keratinocyte behavior, showing the importance of lncRNAs in epidermal biology and skin homeostasis. Multiple strategies have been developed to study lncRNA functions, including RNA interference (RNAi) and CRISPR-based approaches. However, these methods often show limited efficiencies to reach nuclear transcripts or may introduce confounding genomic effects. Antisense oligonucleotides (ASOs) provide a particularly effective technological option by inducing RNase H–mediated degradation of target transcripts and enabling rapid, reversible, and transcript-specific knockdown without any modification of genomic DNA. Together, these considerations highlight the use of ASOs as a powerful and appropriate approach to investigate the functional roles of lncRNAs in epidermal keratinocytes and to better understand their contribution to cell fate regulation in the skin. Here we describe an optimized protocol for ASO transfection in primary human epidermal keratinocytes cells in order to efficiently knock down specific lncRNA targets. The expression level of the transcripts was quantified using a ddPCR quantitative approach. As a proof of concept, we repressed the expression of the lncRNA LINC02154 in keratinocytes using three different ASOs. This ASO-based approach offers a reliable and versatile platform for functional interrogation of nuclear lncRNAs in epidermal keratinocytes and facilitates deeper understanding of lncRNA-mediated regulation of skin cell fate.