The methodologies and protocols essential for establishing and maintaining callus and cell suspension cultures are presented. It covers the initiation and proliferation of calluses from plant explants, detailing practical protocols for media preparation, explant sterilization, and subculturing. The chapter also addresses cell viability and growth in suspension cultures, emphasizing quantitative measurements to monitor cell density, fresh and dry weight, optical density, and cell viability. Additionally, it explores advanced single-cell culture techniques such as Bergmann’s cell plating, filter paper raft-nurse method, and the micro-drop technique, highlighting their application in isolating and cultivating individual plant cells. These protocols aim to optimize culture conditions and facilitate the successful production of secondary metabolites, providing a valuable resource for researchers and practitioners in plant tissue culture and biotechnology.