The Amazonian manatee (Trichechus inunguis) is endemic in the Amazonian basin and is the only exclusively fresh water sirenian. Historically hunted on a large scale, this species is now considered endangered, and studies on the reproductive physiology are critical for the improvement of reproductive management of captive and wild populations of manatees. The aim of this study was to verify the viability of androgen measurement in saliva, lacrimal, urine, and fecal samples of the Amazonian manatee by conducting a hormone challenge. Two adult male manatees (A-1 and A-2) were submitted to an experimentation protocol of 12 day (D1 to D10). On D0, the animals received an intramuscular injection of gonadotropin-releasing hormone (GnRH)-analogue. Salivary, lacrimal, urinary, and fecal samples were collected daily (between 0800 hours and 0900 hours) and frozen at −20°C until assayed. Fecal samples were lyophilized, extracted with 80% methanol, and diluted in buffer before the radioimmunoassay (RIA). Urine samples underwent acid hydrolysis and were diluted in depleted bovine serum. Salivary and lacrimal samples were assayed without the extraction step. Hormonal assays were conducted with a commercial testosterone RIA kit. An androgen peak (>median 2 interquartile range [IQR]) was observed in all matrices of both animals, although it was less prominent in the lacrimal samples of A-2. However, the fecal androgen peak (A-1 peak  =  293.78 ng/g dry feces, median [IQR]  =  143.58 [32.38] ng/g dry feces; A-2 peak  =  686.72 ng/g dry feces, median [IQR]  =  243.82 [193.16] ng/g dry feces) occurred later than urinary (A-1 peak  =  648.16 ng/mg creatinine [Cr], median [IQR]  =  23.88 [30.44] ng/mg Cr; A-2 peak  =  370.44 ng/mg Cr, median [IQR]  =  113.87 [117.73] ng/mg Cr) and salivary (A-1 peak  =  678.89 pg/ml, median [IQR]  =  103.69 [119.86] pg/ml; A-2 peak  =  733.71 pg/ml, median [IQR]  =  262.92 [211.44] pg/ml) androgen peaks. These intervals appear to be correlated with the long digesta passage time in this species. The salivary and urinary peaks were closely associated. These results demonstrate that androgen concentrations in saliva, urine, or feces samples reflect reliably physiologic events and are a powerful tool for noninvasive reproductive monitoring of Amazonian manatees.