The role of cytology in molecular oncology has significantly grown in importance, encompassing not only the identification of primary lesions but also functioning as a guide for detecting recurrences, monitoring patients, and providing guidance for treatment. The main cytological procedures include aspiration and exfoliative cytology, and a diverse array of sample types are utilized, such as cell blocks, liquid-based or centrifugation specimens, and smears. While cell blocks have traditionally been predominant for immunohistochemical (IHC) and molecular studies, the suitability of other cytological specimens for complementary tests has been confirmed. However, the implementation of advanced techniques on cytological samples encounters challenges, necessitating standardized protocols and rigorous validation processes. Dependable outcomes hinge on internal laboratory comparisons and cross-institutional validations. In addition, the pivotal role of on-site pathologists or cytotechnologists during aspiration procedures (ROSE) ensures meticulous sample handling, enabling the execution of advanced analyses, including IHC and molecular markers. To enhance accuracy, fostering collaboration among cytotechnicians, pathologists, and stakeholders is essential. Effective communication augments comprehension and result interpretation. In conclusion, the ascending significance of cytology in molecular oncology demands tailored strategies for technique implementation, protocol standardization, and enhanced collaboration, ultimately promising to revolutionize cancer diagnostics and treatment approaches. This chapter explores the fundamental technical principles of cytological methods, examines the primary applications and limitations of performing IHC and molecular studies in cytology, and discusses the challenges and improvements for enhancing the reliability and applications of cytology in personalized medicine.