Approximately 80-96% of people with amyotrophic lateral sclerosis (ALS) become unable to speak during the disease progression. Assessing upper and lower motor neuron impairment in bulbar regions of ALS patients remains challenging, particularly in distinguishing spastic and flaccid dysarthria. This study aimed to evaluate acoustic voice parameters as useful biomarkers to discriminate ALS clinical phenotypes. Triangular vowel space area (tVSA), alternating motion rates (AMRs), and sequential motion rates (SMRs) were analyzed in 36 ALS patients and 20 sex/age-matched healthy controls (HCs). tVSA, AMR, and SMR values significantly differed between ALS and HCs, and between ALS with prevalent upper (pUMN) and lower motor neuron (pLMN) impairment. tVSA showed higher accuracy in discriminating pUMN from pLMN patients. AMR and SMR were significantly lower in patients with bulbar onset than those with spinal onset, both with and without bulbar symptoms. Furthermore, these values were also lower in patients with spinal onset associated with bulbar symptoms than in those with spinal onset alone. Additionally, AMR and SMR values correlated with the degree of dysphagia. Acoustic voice analysis may be considered a useful prognostic tool to differentiate spastic and flaccid dysarthria and to assess the degree of bulbar involvement in ALS.