In vivo protocols were developed for prescreening of rhizobacteria for the ability to reduce damage to cotton seedlings induced by Fusarium oxysporum, Pythium ultimum, and Thielaviopsis basicola. In radicle and hypocotyl assays, growth medium, form of inoculum, and host tissue age at inoculation were evaluated for each pathogen for best symptom development. Pathogens grown on potato dextrose agar induced relatively higher symptom ratings in both assays. A suspension prepared from one 9-cm culture plate and 50 ml sterile water induced best symptoms with P. ultimum and F. oxysporum in the radicle assay and with all three pathogens in the hypocotyl assay. A 10^-1 dilution of the suspension provided best symptom expression by T. basicola in the radicle assay. Inoculation of radicles at germination and hypocotyls at six days after planting with P. ultimum produced higher symptoms ratings. With T. basicola and F. oxysporum best symptoms development occurred when one-day old radicles and hypocotyls at 6 and 12 days after planting were inoculated, respectively. Due to subnormal symptom development in the hypocotyl assay, only the radicle assay was selected for validation of antagonistic activities of selected biocontrol rhizobacteria. Using this protocol, the ability of Bacillus subtilis (Kodak isolate) and Pseudomonas fluorescens Dagger G isolate) to reduce damage from P. ultimum, F. oxysporum, and T. basicola was demonstrated.