Antibody-based therapeutics and recombinant protein reagents are often produced in mammalian expression systems, which provide human-like post-translational modifications. Among the available mammalian cell lines used for recombinant protein expression, Chinese hamster ovary (CHO)-derived suspension cells are generally utilized because they are easy to culture and tend to produce proteins in high yield. However, some proteins purified from CHO cell overexpression suffer from clipping and display undesired non-human post translational modifications (PTMs). In addition, CHO cell lines are often not suitable for producing proteins with many glycosylation motifs for structural biology studies, as N-linked glycosylation of proteins poses challenges for structure determination by X-ray crystallography. Hence, alternative and complementary cell lines are required to address these issues. Here, we present a robust method for expressing proteins in human embryonic kidney 293 (HEK293)-derived stable pools, leading to recombinant protein products with much less clipped species compared to those expressed in CHO cells and with higher yield compared to those expressed in transiently-transfected HEK293 cells. Importantly, the stable pool generation protocol is also applicable to HEK293S GnTI-(N-acetylglucosaminyltransferase I–negative) and Expi293F GnTI-suspension cells, facilitating production of high yields of proteins with less complex glycans for use in structural biology projects. Compared to HEK293S GnTI-stable pools, Expi293F GnTI-stable pools consistently produce proteins with similar or higher expression levels. HEK293-derived stable pools can lead to a significant cost reduction and greatly promote the production of high-quality proteins for diverse research projects.