We have developed an automated cell counting method that uses images obtained at multiple focal heights to enumerate cells in confluent culture. By taking the derivative of image intensity with respect to focal height using two complementary images, we are able to count high-density monolayers of cells over a large image area. Our method resists errors arising from variability in the focal plane caused by flatness or tilt non-uniformities with a minimal amount of focal plane alignment, allowing the automated collection of images across a large area. Biotechnol. Bioeng. © 2013 Wiley Periodicals, Inc.