To investigate the effect of number and combination of b values used on the accuracy of estimated Intravoxel Incoherent Motion (IVIM) parameters using simulation and clinical data.Simulations with seven combinations of b values were performed for 4, 6, 8, and 13 numbers of b values with six different values of D, D*, and f parameters. Two methodologies were implemented for IVIM analysis: standard biexponential model (BE) and biexponential model with total variation penalty function (BE + TV). Clinical data set of six patients with prostate cancer was retrospectively analyzed using 4, 8, and 13 b values.BE + TV method showed lesser error and lower variability in simulation and clinical data, respectively. 8 and 13 b values showed good agreement in the values of parameters estimated with high correlation coefficient (ρ = 0.83-0.93). Clinical data showed high spurious noise with lower b values [4 b values leading to high coefficient of variation (CV); however, substantially, lower CV was observed with 8 and 13 b values].BE model with TV penalty function is robust to combination of b values used for IVIM analysis. Combination of 8 b values provided a reasonably good accuracy in IVIM parameters.