Precision medicine in oncology increasingly relies on comprehensive molecular profiling, necessitating adequate biological material for both morphological and molecular analysis. In patients with advanced-stage malignancies, where surgical biopsies are often invasive or unfeasible, cytological samples frequently represent the only accessible material for molecular testing, including next-generation sequencing (NGS). Various cytological preparations, such as direct smears, liquid-based cytology, cytospin preparations, cell blocks, and supernatants, differ in nucleic acid quality and quantity, impacting NGS performance. Despite challenges related to limited cellularity, nucleic acid degradation, and lack of tissue architecture, advances in NGS technology have enabled reliable genomic profiling from minimal cytological input. Cytology thus offers a minimally invasive, rapid, and cost-effective approach for molecular diagnostics and therapeutic decision-making in oncology. However, standardized protocols and rigorous validation are essential to optimize workflows and facilitate the integration of cytology-based NGS into routine clinical practice.