Pancreatic masses pose a diagnostic difficulty due to the technical complexities related to tissue acquisition. Endoscopic ultrasound (EUS)-guided tissue acquisition has transformed the field by allowing access to pancreatic lesions through fine-needle and biopsy. However, diagnostic accuracy differs based on tumor characteristics and procedural factors. This narrative review explores the nuances of tissue acquisition methods for pancreatic tumors, including factors such as tumor location, size, histological characteristics, and needle selection. It assesses the efficacy of different needle designs and maneuvers, including suction techniques and needle passes. Moreover, the diverse tissue preparation methods, including cytological smear, cell block, and direct histology, are discussed, highlighting the importance of tailored approaches based on tumor characteristics. Additionally, the roles of macroscopic on-site evaluation and rapid on-site evaluation in optimizing specimen adequacy are investigated. Furthermore, percutaneous ultrasound-guided biopsy is considered an alternative approach, particularly in settings where EUS is impractical. Additionally, the review emphasizes the emerging trend of using tissue for genetic testing and molecular analysis, requiring high-quality sample acquisition. Future directions in tissue acquisition techniques and their integration into clinical practice are discussed, providing promising avenues for pancreatic disease diagnosis and treatment.