Automated software improves the accuracy and reliability of blood velocity, vessel diameter, blood flow, and shear rate ultrasound measurements, but existing software offer limited flexibility to customize and validate analyses. We developed FloWave.US - an open source software to automate ultrasound blood flow analysis - and demonstrated the validity of its blood velocity (aggregate relative error: 4.32%) and vessel diameter (0.31%) measures with a skeletal muscle ultrasound flow phantom. Compared with a commercial, manual analysis software, FloWave.US produced equivalent in vivo cardiac cycle time averaged mean (TAMean) velocities at rest and following a 10-s muscle contraction (mean bias less than 1 pixel for both conditions). Automated analysis of ultrasound blood flow data was 9.8 times faster than the manual method. Finally, a case study of a lower extremity muscle contraction experiment highlighted the ability of FloWave.US to measure small fluctuations in TAMean velocity, vessel diameter, and mean blood flow at specific time points in the cardiac cycle. In summary, the collective features of our newly designed software - accuracy, reliability, reduced processing time, cost-effectiveness, and flexibility - offer advantages over existing proprietary options. Further, public distribution of FloWave.US allows researchers to easily access and customize code to adapt ultrasound blood flow analysis to a variety of vascular physiology applications.