Agitated saline microbubbles (MBs) is a common contrast agent for determining right-to-left shunt (RLS) by the contrast Transcranial Doppler (c-TCD). The size of the generated bubbles is not standardized in clinical practice. The microbubbles were generated using the recommended manual method by reciprocating motion through two syringes. The bubble size distributions (BSD) were measured using the microscopic shadow imaging technique. The results show that the diameter of MBs is mainly distributed between 10-100 µm, the mean bubble size is between 21-34 μm, the Sauter mean diameter (D 32) is mainly between 50-300 μm, and the standard deviation (SD) is between 6-17 μm in 60 experiments. It provides a more accurate basis for the recommended manual method instability. The high variance values of the BSD indicate that the manual method has low stability and repeatability. The results of this study can be useful for further improvement of the reliability of c-TCD in detecting RLS. Research Highlights This study provided the first detailed descriptions of the size distribution of microbubbles in a flowing contrast agent by the microscopic shadow imaging technique. It reveals significant differences in the bubble size of manual foaming during repeated manipulations for each individual and between individuals.