Unlike normal tissues, tumors have hypoxic regions. Mathematical models suggest that cell survival in tumors is strongly dependent on and limited by the availability of oxygen, though no experimental evidence exists that eliminates the effect of other nutrient gradients and the acidic conditions that exist within the tumor. Our aim was to measure the rate and location of cell death and track cell migration in tumor tissue growing in a spatially and temporally defined oxygen microenvironment, to quantify the effect of modified oxygen gradients in tumors. We have developed an innovative microtechnology that allows spatial as well as temporal control over local oxygen delivery to in-vitro tissues. The gradient generating device consists of a single or an array of noble metal microelectrodes patterned over a glass substrate within a polydimethylsiloxane (PDMS) microchannel, which is filled with an electrolyte (deionized water). Electrolysis generates dissolved oxygen and can be used to generate a user defined oxygen gradient over the cell culture area. A single circular electrode of 10μm diameter was used to generate oxygen. Cylindroids, which were formed by compressing multicellular spheroids in between two parallel surfaces, were positioned over the cell culture area so that their centers coincided with the electrode. A direct correlation between the electrolysis current and the oxygen generation rate was developed. Generation of oxygen at the center of cylindroids created oxygenated regions in otherwise hypoxic regions of the cylindroids. Fluorescent viability stains were used to determine viability profiles and viable regions were observed at the center of the cylindroids when oxygen was supplied for 60 hours. The corresponding controls had necrotic centers. Fluorescent nuclear stains were used to track cell migration and velocity profiles of the migrating cells were obtained. The nuclear morphology was monitored to determine the rate and location of apoptosing cells. A mathematical model for cylindroid growth was developed incorporating chemotaxis of individual cells towards a higher oxygen concentration. Experimental data was used to tune the parameters of the model to predict the relative contribution of chemotaxis (active) and growth (passive) to cell migration in tumor tissue. These experiments demonstrate the ability to study cell response in a spatially and temporally controlled oxygen microenvironment and could be used to predict microenvironment-specific tumor response.