The TIMSTOF Ultra2 is a dedicated instrument for sub-nanogram and single-cell proteomics. It is coupled to an EvoSep One system for rapid semi-automated cleanup and injection of up to 80 unlabeled single cells per calendar day. When employing multiplexed reagents the system can analyze up to 700 single cells per day after suitable inter- and intra-batch quality control and assurance standards. Single-cell isolation and digestion can be performed using a Tecan Uno system for less complex mixtures of dissociated cells. Single cells can also be isolated and pre-processed using our provided protocol for delivery or shipment to the core for analysis.