Azoospermia is a common finding in male alpacas which present for infertility. The challenge is to differentiate azoospermia of testicular origin from non-testicular origin. In several species, alkaline phosphatase (AP) concentrations in seminal plasma have been used as a diagnostic marker of contributions of the testis and epididymis to the ejaculate. The purpose of this study was to determine whether AP assay could differentiate testicular from non-testicular azoospermia in male alpacas. An experimental model of bilateral outflow obstruction (pre-scrotal vasectomy) was used in 22 male alpacas, aged 2–9 years. No reproductive history was available. Animals were submitted for electroejaculation (EE) under general anaesthesia and vasectomy performed. Five weeks later, animals were submitted for EE. Vasectomy was not successful in one animal, which was removed from analysis. AP levels were compared in seminal plasma in the pre- and post-vasectomy samples. The mean ± SEM concentration of AP in pre-vasectomy seminal plasma was 504.29 ± 166.45 U/l (range 10–2910); the post-vasectomy levels were 252.48 ± 81.77 U/l (range 0–1640; p = 0.06). In 71.4% of animals, AP levels decreased, varying from 18% to 100% reduction. Results of this study suggest that AP is not produced exclusively by the testis and epididymis in alpacas and that AP assay is not a valid diagnostic test for determination of origin of azoospermia; the gold standard for diagnosis of origin of azoospermia remains testicular biopsy.