Antisense oligonucleotides (ASOs) are a powerful class of therapeutics designed to target RNA sequences and regulate gene expression, offering promising treatment avenues for various genetic disorders, neurodegenerative diseases, cancers, and viral infections. The bioanalysis of ASOs presents unique challenges due to their distinct chemical and biological characteristics, requiring specialized analytical methods and regulatory oversight. This review explores the latest methodologies in ASO bioanalysis, assesses the current regulatory environment, and considers future directions, particularly in the context of critical and emerging technologies (CETs) relevant to national security strategies. ASOs, classified under biotechnology, play a crucial role in gene expression modulation and protein regulation. By integrating insights from CETs, this review emphasizes the strategic significance of ASO bioanalysis in driving advancements in medical science and contributing to national security.