Alpacas, like other camelids, are induced ovulators. A study was designed to determine the effect of the ovulation-inducing stimulus on the biochemical composition of follicular fluid. Adult female alpacas (n = 18) were examined daily for 3 days by transrectal ultrasonography using a 5-MHz linear-array transducer (Aloka SSD-500, Tokyo, Japan). When the largest growing ovarian follicle was ≥7 mm, alpacas were given 1.0 mL of seminal plasma intramuscularly (SP, n = 9) or 40 µg of busereline acetate intramuscularly (GnRH, n = 9). A transvaginal transducer with an attached needle guide (Aloka UST-945BP-5) was used for collection of follicular fluid 22 h post-induction. Follicular contents were then centrifuged at 800 × g for 20 min to separate the fluid from the cells. The follicular fluid was collected and stored at –20°C until analysis with a semi-automatic biochemical analyzer (SINOWA, China). The results were glucose 49.17 and 47.95 (mg/dL; P > 0.05), total protein 1.85 and 1.15 (g/dL; P < 0.05), albumin 1.11 and 1.13 (g/dL; P > 0.05), triglycerides 3.94 and 3.16 (mg/dL; P > 0.05), cholesterol 39.01 and 42.5 (mg/dL; P > 0.05), phosphatase 32.68 and 21.36 (IU/L; P < 0.05), alanine aminotransferase 3.66 and 5.07 (IU/L; P > 0.05), and lactate dehydrogenase 42.17 and 27.27 (IU/L; P > 0.05) for SP or GnRH treatments, respectively. Results suggest the need to continue research to explain the effect of possible differences in total protein, cholesterol, and phosphatase on oocyte-expressed genes and follicular development.