Chondrichthyans are one of the most vulnerable groups of marine species, with increasing numbers being listed as endangered or threatened. Fundamental to the conservation and management of any animal is an understanding of their reproduction and particularly, the size at which individuals enter the reproductive population. Size at maturity in sharks is typically obtained by macroscopic examination of the gonads after sacrificing the fish. Using the draughtboard shark Cephaloscyllium laticeps as a case study, we found that equivalent information can be obtained non-destructively using steroid hormone levels measured from a blood sample. This technique allowed us to release the shark after only 1 to 2 min of handling. A combination of plasma steroid hormone concentrations and an external measurement (total length for females and clasper length for males) was used to determine that over 90% of female and 95% of male sharks were either juveniles or adults. The estimates of size at maturity from the hormone analysis were within 3% of equivalent values of dissected sharks. Once validated, hormone levels can also be used to determine seasonal reproductive patterns for the adult population. Our results demonstrated how measurement of plasma levels of steroid hormones can provide a non-destructive method for obtaining reproductive data necessary for managing vulnerable and endangered shark species. The development of a non-destructive sampling tool also allows for improved ethical investigation into this vulnerable group of marine fishes.