A procedure based on quantitative real-time PCR was evaluated for the detection and quantification of Bacillus cereus spores. Several methods for DNA isolation, such as various heat treatments and germination solutions, were evaluated on spore suspensions of representative strains of the B. cereus group. Overall, the commercially available DNeasy tissue kit yielded the maximum amount of DNA. The procedure also was used to construct calibration curves for different food matrices, with a wide spore quantification range of 5 log units using serial dilutions of spore suspensions of B. cereus CECT 148T. The detection limit for B. cereus in artificially contaminated liquid egg and reconstituted infant formula was about 4 spores per reaction or 60 spores per ml. The newly developed methodology based on the DNeasy tissue kit and an SYBR Green quantitative real-time PCR assay is very suitable for the rapid and accurate detection and quantification of B. cereus group strains and their spores in food samples.