This study investigates the effects of substrate composition on root architecture, plant growth, and allelopathic secondary metabolites, specifically benzoxazinoids (BXs), in the rhizospheres of rye (Secale cereale L.) and redroot pigweed (Amaranthus retroflexus L.). Given the complexities of root exudate analysis, including the influence of substrate on root morphology and exudation, the experiment compared plant growth and BX release in two substrates: glass microbeads and a mixture of clay beads and attapulgite. Rye, pigweed, and co-cultures of the two were grown under controlled conditions, with root and shoot parameters measured to assess substrate suitability. Additionally, UPLC-QTOF-MS was used to analyze BXs in rye and rye–pigweed co-cultures. The results demonstrated that the clay bead and attapulgite mixture provided better growth conditions and was effective for BX extraction, making it a suitable substrate for studying allelopathy in controlled environments. The findings highlight the critical role of substrate composition in both plant development and the study of root exudates, with implications for better understanding of crop–weed interactions and allelopathy.