Surface charge is a critical feature of microbes that affects their interactions with other cells and their environment. Because bacterial surface charge is difficult to measure directly, it is typically indirectly inferred through zeta potential measurements. Existing tools to perform such characterization are either costly and ill-suited for non-spherical samples or rely on microfluidic techniques requiring expensive fabrication equipment or specialized facilities. Here, we report the application of commercially available PMMA microfluidic chips and open-source data analysis workflows for facile electrokinetic characterization of particles and cells after prior zeta potential measurement with a Zetasizer for calibration. Our workflows eliminate the need for microchannel fabrication, increase measurement reproducibility, and make zeta potential measurements more accessible. This novel methodology was tested with functionalized 1 μm and 2 μm polystyrene beads as well as Escherichia coli MG1655 strain. Measured zeta potentials for these samples were in agreement with literature values obtained by conventional measurement methods. Taken together, our data demonstrate the power of this workflow to broadly enable critical measurements of particle and bacterial zeta potential for numerous applications.