Arabidopsis thaliana is one of the most widely used model organisms in plant sciences. Because of the increasing knowledge in the understanding of its molecular pathways, a reproducible and stable growth set-up for obtaining uniform plants becomes more important. In order to be able to easily harvest and study both roots and shoots, and to allow simple exposure to water-soluble toxic substances, a hydroponic system is the desired cultivation method for controlled plant growth. Based on earlier developed hydroponic cultivation protocols, a hydroponic set-up was optimized and statistically validated using linear mixed-effects models. In order to determine important components that influence the level of variability in a hydroponic set-up, stress-related indicators were examined at the biochemical as well as at the molecular level. It is highly recommended that statistical as well as biological assumptions are carried out before post-analyses are performed. Therefore, we suggest a model where factors that influence variability such as the usage of different pots and harvesting on different times are taken into account in the analyses. Furthermore, in contrast to what has been reported in earlier studies, our findings indicate that continuous aeration of the hydroponic solution is highly important.