Background and aims <p>A major challenge in root exudation research is obtaining exudates samples that accurately reflect the exudation processes under natural soil growth conditions. Both growth environment and experimental setup can significantly influence root exudation dynamics. This study investigated how different experimental systems and growth conditions affect carbon exudation in maize (<i>Zea mays</i> L.) roots and whether these factors could influence the detection of genotypic differences between the wild type (B73) and its hairless mutant, <i>rth3.</i></p> Methods <p>Maize plants were grown under various experimental conditions, including soil-based and hydroponic systems. Root exudates were collected using a combination of traditional and innovative sampling approaches. Carbon exudation rates were compared across experimental setups and genotypes. Laboratory results were further compared with data from a separate field experiment.</p> Results <p>Exudation rates obtained from soil-based laboratory experiments were comparable to those observed in the field under similar growth temperatures. The contribution of root hairs to total carbon exudation was negligible compared to the effect of growth conditions and experimental setup. Large differences in root biomass introduced bias into exudation measurements, particularly when root to sampling volume ratio (RSVR) varied substantially.</p> Conclusions <p>Experimental setup and environmental conditions have a strong influence on root exudation measurement. Soil-based laboratory systems that closely replicate field conditions, particularly temperature, can serve as reliable proxies for field experiments, providing ecologically meaningful data. Maintaining a consistent RSVR is also essential for obtaining accurate and comparable results. These findings offer important methodological guidance for reliably quantifying root carbon exudation in maize</p>