Muscle volume (MV) is an important parameter for understanding muscle morphology and adaptations to training, growth and pathology. In this study, we assessed the validity of freehand 3D ultrasound (3DUS) for measuring medial gastrocnemius MV in adults, typically developing (TD) children and children with cerebral palsy (CP). We also assessed the validity between our direct measures of MV and estimates derived from anatomical cross sectional area (ACSA) and muscle length (ML), using previously outlined methods. The medial gastrocnemius of all groups was scanned with 3DUS and MRI. Images from both methods were digitized to derive MV, ACSA and ML. Measured MV was compared between methods and compared to estimated MV derived from recently published algorithms. MV had a mean difference of −0.13% (standard error of estimate (SEE) = 2.23%, R 2 = 0.99) between MRI and 3DUS and 19.82% (SEE = 4.73% and R 2 = 0.99) and −3.11% (SEE = 6.55%, R 2 = 0.99) mean differences between the measured and estimated MV from two methods of estimation. The 3DUS is a valid method for the measurement of MV in adults, TD children and those with CP. Estimation methods of MV may be useful in clinical practice, but require further replication on various populations and careful methodological consideration.