The coupling of microfluidic devices with surface plasmon resonance imaging (SPRI) has emerged in recent years as a novel approach for the simultaneous monitoring of interactions of biomolecules arrayed onto gold Substrates. In order to minimize a variety of effects which affect the final determination of kinetic parameters (non-specific interactions above all), difficult choices of appropriate references are often encountered in carrying out SPRI investigations. A common solution to these problems consists of laborious experimental setup involving the use of specially designed microchannels and tedious manipulation of the gold substrate that often produces surface degradation. In this work, a discussion about appropriate choice of references in SPRI measurements is opened and the use of alternative microfluidic patterns coupled to the SPRI system is proposed as a solution to the above mentioned problems. Specifically, a Y-shaped SPRI flow cell has been constructed from masters in polyvinyl chloride and it has been identified as one of the most suitable experimental approach for obtaining appropriate referencing during SPRI experiments. The experimental set up has been tested in a real time study of the interaction between the Datura Stramonium Agglutinin and the asialofetuin and the obtained results demonstrate the suitability of such microfluidic network in SPRI investigations. (C) 2009 Elsevier B.V. All rights reserved.