We introduce the ratio of nonflocculent versus total biomass as a criterion for starting cell separation from the medium. This criterion can be applied for the automation of the process regardless of the process dynamics. Its minimum indicates the optimum period of time for the start of the separation process with regard not only to nonflocculent cell concentration, but also medium attributes. In contrast to the concentration of nonflocculent cells, which has two minima, first at the beginning of the process and another broader one in the period during which maximum flocculation is present, the ratio has a single minimum and can therefore be implemented as a criterion for cell separation. To calculate the ratio value, in addition to an on-line method for nonflocculent biomass measurement described elsewhere, an on-line method for the total biomass of flocculent yeast is proposed. It is based on the absorbency measurement of the cell biomass, previously deflocculated by EDTA. Therefore, it can be applied in bioprocesses with transparent media and yeast that can be deflocculated by EDTA.