One of the key early season diseases of cotton is Fusarium wilt caused by Fusarium oxysporum f. sp. vasinfectum (Fov). Recent emergence of highly virulent Fov race 4 (Fov4) and its aggressiveness toward Gossypium barbadense (pima) cultivars are raising significant concerns for the US cotton industry. One of the key challenges in studying Fov4 virulence and cotton Fusarium wilt pathogenesis is establishing a disease assay strategy that can help researcher overcome several technical challenges, including efficient infection and highly reproducible and consistent symptom development. Here, we have developed a small-scale, soil-free Fusarium wilt disease assay that can complement conventional assays with faster symptom development and high reproducibility in infected pima cotton seedlings. Our data showed statistically significant differences (p<0.0001) between Fov4-infected and non-infected pima cotton at 4 and 6 days post inoculation (dpi) when compared to control experiments. At 6 dpi, longitudinal observations under magnification showed Fov4 colonization in primary xylem of infected plants, which is a common symptom observed in Fov4 triggered Fusarium wilt in pima cotton. While this is an artificial assay system, this soil-free disease testing strategy can offer another strategy to supplement current assays when studying pathogen-host interaction in soil-borne diseases.