Human embryonic kidney cells (HEK293) are a widespread choice for recombinant protein expression. To optimise yields, the hydrolysate Tryptone N1 (TN1) is commonly added post-transfection. TN1 is obtained by controlled enzymatic digestion of casein. As an animal by-product, TN1 faces strict regulations during cross-country shipments as compared to plant-based products. This raises the question whether plant-derived peptides are a suitable alternative to TN1. Using polyethyleneimine as a cationic polymer, we transfected HEK293-6E cells grown in suspension in serum-free medium and divided the transfectants into 4 groups. Two plant-based hydrolysates derived from pea and broad-bean, respectively, were compared with TN1 and a no-hydrolysate control group. Transfection efficiency between biological replicates, as determined by transfection of a GFP-encoding plasmid, was similar. We monitored the cultures for total cell numbers and viability at days 1, 4 and 5 post-transfection. Both plant-based hydrolysates and TN1 showed similar live cell percentages, in contrast to the no-hydrolysate control, which showed lower viability. Five days post-transfection. Expressed protein was retrieved from the serum-free culture supernatant and quantified through Western blotting using stain-free technology and total lane quantification. The plant-derived pea and broad-bean hydrolysates resulted in similar expression quantity as animal-derived TN1; all three hydrolysates were better than no hydrolysate. We conclude that plant-derived hydrolysates are a suitable replacement for TN1.