AbstractCompensating in flow cytometry is an unavoidable challenge in the data analysis of fluorescence-based flow cytometry. Even the advent of spectral cytometry cannot circumvent the spillover problem, with spectral unmixing an intrinsic part of such systems. The calculation of spillover coefficients from single-color controls has remained essentially unchanged since its inception, and is increasingly limited in its ability to deal with high-parameter flow cytometry. Here, we present AutoSpill, an alternative method for calculating spillover coefficients. The approach combines automated gating of cells, calculation of an initial spillover matrix based on robust linear regression, and iterative refinement to reduce error. Moreover, autofluorescence can be compensated out, by processing it as an endogenous dye in an unstained control. AutoSpill uses single-color controls and is compatible with common flow cytometry software. AutoSpill allows simpler and more robust workflows, while reducing the magnitude of compensation errors in high-parameter flow cytometry.