<p>The overexploitation of horseshoe crab species, including the American horseshoe crab, <i>Limulus polyphemus,</i> and Tri-spine horseshoe crab, <i>Tachypleus tridentatus</i>, for their amoebocyte lysate in biomedical applications is a well-documented issue. Similar concerns arise for coastal horseshoe crab, <i>Tachypleus gigas</i>, where biomedical bleeding may pose significant ecological and reproductive risks. This study investigates the protein profile of <i>T. gigas</i> lysate and examines the effects of hemolymph extraction on their pairing and spawning behaviors. Hemolymph extractions were conducted in three stages: the first extraction occurred before pairing and spawning trials, followed by the second and third extractions at subsequent intervals. Results showed a significant decline in lysate protein concentration after the third extraction. While band intensity at various molecular weights differed significantly, no notable variation was observed between males and females throughout the extraction series. Interestingly, only bled males successfully paired and spawned with available females, whereas no males selected bled females for pairing. These findings suggest that hemolymph extraction disrupts the reproductive behavior of <i>T. gigas</i>, particularly in females. This study provides crucial baseline data to inform sustainable management plans for <i>T. gigas</i> populations utilized in the biomedical industry.</p>