Microfluidic platforms are ideal for generating dynamic temporal and spatial perturbations in extracellular environments. Single cells and organisms can be trapped and maintained in microfluidic platforms for long periods of time while their responses to stimuli are measured using appropriate fluorescence reporters and time-lapse microscopy. Such platforms have been used to study problems as diverse as C. elegans olfaction (Chronis et al. Nature Methods 4:727-731, 2007), cancer cell migration (Huang et al. Biomicrofluidics 5:13412, 2011), and E. coli chemotaxis (Ahmed et al. Integr Biol 2:604-629, 2010). In this paper we describe how to construct and use a microfluidic chip to study the response of single yeast cells to dynamic perturbations of their fluid environment. The method involves creation of a photoresist master mold followed by subsequent creation of a polydimethylsiloxane (PDMS) microfluidic chip for maintaining live yeast cells in a channel with two inputs for stimulating the cells. We emphasize simplicity and the methods discussed here are accessible to the average biological laboratory. We cover the basic toolbox for making microfluidic lab-on-a-chip devices, and the techniques discussed serve as a starting point for creating sophisticated microfluidic devices capable of implementing more complicated experimental protocols.