Current techniques for characterizing biofilm physiology lack the signal filtering capability required for quantifying signals associated with real time biologically active transport. Though a great deal was learned from previous investigations, no results have been reported on the characterization of in vivo, real time biofilm flux Using non-invasive (non-destructive) techniques. This article introduces the self-referencing technique for applications in biofilm physiology. Self-referencing is a non-invasive sensing modality which is capable of sensing changes in biologically active analyte flux as small as 10 fmol cm(-2) s(-1). Studies directly characterizing flux, as opposed to concentration, have the advantage of quantifying real time changes in biologically active transport which are otherwise lost to background noise. The use of this modality for characterizing biofilm physiology is validated with a reversible enzyme inhibition study. The experiment used self-referencing potentiometric sensors for quantifying real time ammonium and nitrite flux. Amperometric and optical sensing methods, though not presented herein, are also powerful sensing tools which benefit from operation in self-referencing mode. Reversible ammonia monooxygenase inhibition by a copper chelator (thiourea), and Subsequent relief by excess copper addition was successfully demonstrated using self-referencing ion-selective microelectrodes for a mature Nitrosomonas europaea biofilm.