This paper presents a technique for accurate estimation of growth in root culture systems. Biomass correlations, were used to estimate fresh weight time course data in shake flasks and reactors based on a model of liquid nutrient uptake and osmolality, to account for changing specific water content of roots. This mass balance technique has been developed to permit accurate aseptic on-line estimation of dry weight (DW), fresh weight (FW), and liquid volume (V) in root cultures utilizing either refractive index or electrical conductivity of the liquid medium along with liquid medium osmolality. The ability to predict fresh weight is particularly important since this is proportional to the biomass volume fraction which determines mass transfer and other culture transport characteristics. The proposed model has been validated with time course information (DW, FW, and V) from 125 mt shake flasks and corroborated with data obtained from 2 L reactors.