The combination of automatic image acquisition and automatic image analysis of premature chromosome condensation (PCC) spreads was tested as a rapid biodosimeter protocol. Human peripheral lymphocytes were irradiated with 60Co gamma rays in a single dose of between 1 and 20 Gy, stimulated with phytohaemaglutinin and incubated for 48 h, division blocked with Colcemid, and PCC-induced by Calyculin A. Images of chromosome spreads were captured and analysed automatically by combining the Metafer 4 and CellProfiler platforms. Automatic measurement of chromosome lengths allows the calculation of the length ratio (LR) of the longest and the shortest piece that can be used for dose estimation since this ratio is correlated with ionizing radiation dose. The LR of the longest and the shortest chromosome pieces showed the best goodness-of-fit to a linear model in the dose interval tested. The application of the automatic analysis increases the potential use of the PCC method for triage in the event of massive radiation causalities. © 2014 The Author.