Producing increasingly complex proteins with higher yields and higher throughput is critical to accelerating breakthroughs in protein research and therapeutic drug development. Here, we introduce the Expi293 PRO Expression System, a next-generation transient expression system developed to address the unmet needs of protein expression scientists. The Expi293 PRO Expression System comprises: 1) a new, clonally-derived HEK293 cell line capable of producing difficult to express proteins and supporting milliliter to multi-liter scale protein production, 2) optimized culture medium and transfection reagent to enable high density cell transfection, 3) components and protocols designed for easy incorporation into automated workflows, and 4) a streamlined protocol with the fewest steps possible to minimize hands-on time and maximize the number of transfections that can be performed in a workweek. Together, these optimized components enable the Expi293 PRO Expression System to produce multi-fold improvements in protein yields across a broad variety of proteins compared to existing 293 expression systems with protein titers up to 4 grams per liter, making the Expi293 PRO Expression System an ideal next-generation offering to meet the ever-expanding needs of protein expression scientists.