The collection of alpaca semen presents difficulties because of the characteristics of mounts and copulation time in addition to the high viscosity of semen. The biophysical and biochemical characteristics of alpaca semen were investigated. Semen samples from four adult males from 6 to 7 years of age were collected using an electroejaculation technique. The biophysical parameters measured in seminal plasma included volume, color, sperm concentration, motility, and viscosity, and the biochemical parameters included pH and glucose, calcium, triglyceride, cholesterol, high-density lipoprotein cholesterol, total protein, albumin, alanine transaminase, alkaline phosphatase, and γ-glutamyltranspeptidase concentrations. The mean time for semen collection was 10.3 ± 1.5 min; the volume varied from 1.2 to 3.8 and the colour from clear to milky, and pH was 7.4 ± 0.03. Mean concentration of spermatozoa was 69.3 ± 193 million mL–1, and motility was 52.4 ± 9.7%. Biochemical parameters were glucose (mg dL–1) 8.22 ± 0.77; cholesterol (mg dL–1) 79.78 ± 5.64; triglycerides (mg dL–1) 44.12 ± 7.38; high-density lipoprotein cholesterol (mg dL–1) 4.73 ± 0.30; total protein (g dL–1) 2.36 ± 0.15; albumin (g dL–1) 0.97 ± 0.33; calcium (mg dL–1) 11.77 ± 1.74; alanine transaminase (U L–1) 17.92 ± 9.09; alkaline phosphatase (U L–1) 288.76 ± 279.59; and γ-glutamyltranspeptidase (U L–1) 89.62 ± 39.09. These results provide information on the biophysical and biochemical characteristics of alpaca semen collected by electroejaculation, with variable results in the enzymatic components.