Southern hairy-nosed wombats (SHNW) do not breed well in captivity and the collection of data to accurately evaluate and characterise the female reproductive cycle has been limited. The aim of this study was to biologically validate an enzyme-immunoassay (EIA) for the detection and analysis of luteinizing hormone (LH) in non-invasive urine samples. During the 2013 breeding season, four anaesthetised female SHNW received 4 µg exogenous gonadotrophin releasing hormone (GnRH, Burserelin: Intervet). Serial blood and urine samples were collected immediately before and up to three, or 72 hours respectively, post GnRH injection. All serum and urine samples were stored frozen (-20̊C) until EIA hormone analysis of LH could be conducted. EIA analysis confirmed no serum LH response to 4 µg of exogenous GnRH. The trial was attempted again during the 2014 breeding season. Four females received a single 8 µg GnRH injection and two of those females received an additional 2 µg GnRH thirty minutes following the initial dose. All females responded to the higher dose. Mean baseline serum LH was 1.2 ± 0.18 ng/mL and peaked between 1.2 and four fold ten to 17 minutes post GnRH injection (peak range 1.9 – 4.2 ng/mL). Mean baseline urinary LH was 0.064 ± 0.0101 ng/mg creatinine. LH peaked at 2.2 to 2.7 fold between two and three hours post GnRH injection. This is the first study to successfully detect and evaluate LH concentrations in urine samples in a marsupial species. The assessment of longitudinal urine samples for LH concentration will provide valuable information regarding LH metabolism and regulation, and the timing of ovulation in relation to mating and progesterone secretion, increasing our knowledge of the reproductive biology of SHNW. Further, the difference in GnRH dose rate responses may be an important consideration when developing assisted reproductive technologies for SHNW.