Seed treatment is widely adopted in agriculture due to its effectiveness in protecting seeds against pathogens and insect pests during early crop development. However, questions remain regarding the effects of the interval between seed treatment and sowing, and the suitability of substrates used in germination tests. This study evaluated how different substrates and short storage periods after chemical treatment affect the physiological quality of soybean seeds treated with thiamethoxam, metalaxyl-M, and fludioxonil. The experiment used a trifactorial scheme, considering the presence or absence of seed treatment, four periods between treatment and test installation (0, 2, 4, and 6 days), and different substrates for the germination test (germitest paper, sand, and vermiculite, individually and in combination). The analyzed variables included germination percentage, first germination count, shoot length, root length, total seedling length, number of roots, and total dry mass. Results indicated that short storage periods after seed treatment did not significantly affect germination and vigor, and substrate type had minimal influence on physiological quality. Although vermiculite promoted slightly greater seedling development, this did not warrant its routine use over traditional substrates. In conclusion, short storage periods after chemical treatment do not compromise the physiological quality of soybean seeds, and germitest paper remains an appropriate substrate for germination tests with treated seeds.