Background: The aim of this study was to cross-validate functional transcranial Doppler (FTCD) and brain single photon emission computed tomography (SPECT) using complex activation paradigm and to test the feasibility of FTCD in complex neuroactivation research. Methodology: The study was performed in the group of 60 healthy, right-handed subjects. Cerebral blood flow velocity (CBFV) was measured in both middle cerebral arteries (MCA) during baseline and during computer game interaction, using FTCD. Identical stimulus and response patterns were used in the subgroup of 15 subjects that underwent brain SPECT. Quantitative assessment of results was done to detect the percentage variation between the two measurements. Results: A statistically significant increase of cerebral blood flow (CBF) was detected by FTCD and brain SPECT (p<0.0001). In comparison between the two methods, statistically significant association (r=0.5608, P=0.030, coefficient of determination r2 = 0.3154) was detected only for CBFV increase in the right MCA and for the rightsided cerebral blood perfusion (CBP) increase, indicating that these two methods only partially measure the same characteristics associated with activation of specific brain areas. Conclusion: In comparison with SPECT, FTCD is not sufficiently sensitive method for evaluation of CBF changes during complex activation paradigm. Our study represents negative evidence and stand against the common belief that FTCD is as good as other neuroimaging methods used for CBF measurements during neuroactivation. Therefore, FTCD might not be a reliable and suitable method for evaluation of CBF changes during complex neuroactivation paradigm.