Non-invasive hormone monitoring of endangered wildlife provides an essential tool to optimize breeding strategies, mainly in captive management. However, the protocol of reproductive hormone monitoring on pregnancy status using non-invasive faecal samples in Malayan tapir is still inconclusive. Therefore, we compared the metabolites reproductive hormones; 17β-oestradiol and progesterone in the faecal samples extracted using methods of Schwarzenberger et al. (1996) (Methods A and B), Brown et al. (2001) (Method C) and Shutt et al. (2012) (Method D) from pregnant and nonpregnant captive Malayan Tapir. Faecal samples from four female of Malayan Tapir (Tapirus indicus) were collected for five months at Sungai Dusun Wildlife Reserve (n = 2), Zoo Taiping (n = 1) and Zoo Negara (n = 1), Malaysia. Analysis by liquid chromatography-mass spectrometry (LCMS) confirmed that progesterone was detected in all fecal sample extracted by extraction Method A, while 17β-oestradiol was undetectable in all methods. The measurement of reproductive hormones for pregnancy status via Enzyme-Linked Immunosorbent Assay (ELISA) analysis showed no significant difference (p > 0.05) for progesterone between the fecal extraction methods. Yet the data obtained were not able to validate the pregnancy status, due to similarity in concentration for both pregnant and non-pregnant tapirs. While for 17β-oestradiol, significant results were observed in all extraction methods and in pregnancy status (p < 0.05). Method C was found to be the most reliable extraction method (p < 0.05) to indicate pregnancy status. From the results, it showed that solvent used, boiling process and multiphase extraction plays important role in fecal extraction process in Malayan Tapir.