Scanning electrochemical microscopy (SECM) is a powerful non-invasive analytical technique that can be used to investigate single live cells. SECM depth scan mode allows for the generation of 2D current maps through the precise positioning of a biased ultramicroelectrode (UME). As the current and UME coordinates are embedded into each pixel of the image, hundreds of probe approach curves (PACs) can be obtained from a single depth scan. These experimental PACs can be compared to theoretical PACs to allow for investigation of physiological processes, such as membrane permeability and molecular transport Using an impermeable hydrophilic redox agent, ferrocene carboxylate, quantification of cell height and topography are also possible. Two methods were used to assess cellular heights: (1) the traditional method of PAC comparison: where PACs are extracted over the cell and the dish substrate, and the difference between the closest approach distances, quantifies cell height and (2) the fitting of experimental onto simulated PACs where the cell's diameter was fixed and cell height was parameterized, directly quantifying cell heights. 2D-axially symmetric simulation models are typically used to generate these PACs, however, this method limits the experimentalist to a single PAC over the direct center of the cell and another to a flat insulating surface. By advancing towards 3D simulations, PACs to any location over the cell can now be used. Here, we used a 3D simulation model to quantify cellular topography across the cell to confirm elliptical geometry. These simulations also confirmed the probe-to-cell distances through horizontal sweeps, ensuring proper fitting of the vertical PACs. (C) 2016 Elsevier B.V. All rights reserved.