The objective of this study is to assess whether there is evidence of spinal motoneuron loss in paretic muscles of stroke survivors, using an index measurement called motor unit number index (MUNIX). MUNIX, a recently developed novel neurophysiological technique, provides an index proportional to the number of motor units in a muscle, but not necessarily an accurate absolute count. The MUNIX technique was applied to the first dorsal interosseous (FDI) muscle bilaterally in nine stroke subjects. The area and power of the maximum M-wave and the interference pattern electromyogram (EMG) at different contraction levels were used to calculate the MUNIX. A motor unit size index (MUSizelndex) was also calculated using maximum M-wave recording and the MUNIX values. We observed a significant decrease in both maximum M-wave amplitude and MUNIX values in the paretic FDI muscles, as compared with the contralateral muscles. Across all subjects, the maximum M-wave amplitude was 6.4 ± 2.3 mV for the paretic muscles and 9.7 ± 2.0 mV for the contralateral muscles (p <; 0.001). These measurements, in combination with voluntary EMG recordings, resulted in the MUNIX value of 109 ± 53 for the paretic muscles, much lower than the MUNIX value of 153 ± 38 for the contralateral muscles (p <; 0.01). No significant difference was found in MUSizelndex values between the paretic and contralateral muscles. However, the range of MUSizelndex values was slightly wider for paretic muscles (48.8-93.3 μV) than the contralateral muscles (51.7-84.4 μV). The findings from the index measurements provide further evidence of spinal motoneuron loss after a hemispheric brain lesion.