In this study, a double-tube centrifuge method was employed to determine the effects of soil moisture on the bioactivity of cafenstrole, pretilachlor, benfuresate, oxyfluorfen and simetryn. In general, the available herbicide concentration in soil solution (ACSS) showed little change as soil moisture increased for herbicides. The total available herbicide in soil solution (TASS) typically increased as soil moisture increased for all herbicides. The relationship between TASS and % growth rate based on dry weight showed strong linear relationships for both cafenstrole and pretilachlor, with r2 values of 0.95 and 0.84, respectively. Increasing TASS values were consistent with increasing herbicide water solubility, with the exception of the ionizable herbicide simetryn. Plant absorption and % growth rate exhibited a strong linear relationship with TASS. According to the results suggested that TASS was a better predictor of herbicidal bioactivity than ACSS for all herbicides under unsaturated soil moisture conditions.