Genetically modified (GM) crops undergo large scale multi-location field trials to characterize agronomics, composition, and the concentration of newly expressed protein(s) [herein referred to as transgenic protein(s)]. The concentration of transgenic proteins in different plant tissues and across the developmental stages of the plant is considered in the safety assessment of GM crops. Reference or housekeeping proteins are expected to maintain a relatively stable expression pattern in healthy plants given their role in cellular functions. Understanding the effects of genotype, growth stage and location on the concentration of endogenous housekeeping proteins may provide insight into the contribution these factors could have on transgenic protein concentrations in GM crops. The concentrations of three endogenous proteins (actin, elongation factor 1-alpha, and glyceraldehyde 3-phosphate dehydrogenase) were measured in several different maize hybrids grown across multiple field locations over 2 years. Leaf samples were collected from healthy plants at three developmental stages across the growing seasons, and protein concentrations were quantified by indirect enzymelinked immunosorbent assay (ELISA) for each protein. In general, the concentrations of these three endogenous proteins were relatively consistent across hybrid backgrounds, when compared within one growth stage and location (2-26%CV), whereas the concentrations of proteins in the same hybrid and growth stage across different locations were more variable (12-64%CV). In general, the protein concentrations in 2013 and 2014 show similar trends in variability. Some degree of variability in protein concentrations should be expected for both transgenic and endogenous plant-expressed proteins. In the case of GM crops, the potential variation in protein 123Transgenic Res (2018) 27:331-342 https://doi.org/10.1007/s11248-018-0077-y( 0123456789().,-volV) (0123456789().,-volV) concentrations due to location effects is captured in the current model of multi-location field testing.