The relative lack of efficient methods for evaluating antiseptic antiviral activity, together with weaknesses in the existing European Standard (i.e. NF EN 14476+A1), underlines the need to seek a new method which could allow a more precise evaluation of the antiseptic antiviral activity of chemical agents. This protocol is based on an original gel-based filtration method, using "in-house" G-25 and G-10 Sephadex columns. This method allows the neutralization of both the activity and the cytotoxicity of a large range of molecules, according to their molecular size, in only 1min. The viral model used was the human coronavirus (HCoV) 229E chosen for (i) its increasing medical interest, (ii) its potential resistance and (iii) its representing enveloped viruses mentioned in the European Standard. First, the protocol was validated and it was demonstrated that it was fully operational for evaluating antiviral antiseptic potentiality and useful to screen potentially antiseptic molecules. Second, chlorhexidine (CHX) and hexamidine (HXM) were assessed for their potential anti-HCoV 229E antiseptic activities. It was demonstrated clearly that (i) HXM had no activity on the HCoV 229E and (ii) CHX showed a moderate anti-HCoV 229E activity but insufficient to be antiseptic.