Abstract: The study was aimed to determine the biochemical changes of total protein, glucose, lactate dehydrogenase (LDH) and total lipid in the cryopreserved sperm of mud spiny lobster, Panulirus polyphagus. The mean sperm viability, total protein, glucose, LDH and lipid for control and cryopreserved sperm that stored in liquid nitrogen from control, 6, 12, 24 h, 7, 30 and 60 days were 94.91±2.17% (control), 80.58±1.33, 77.08±2.53, 76.49±3.10, 63.32±7.08, 62.93±3.72 and 60.91±10.78% (sperm viability), 21.44±3.52, 18.82±0.23, 17.79±0.80, 16.98±0.48, 16.65±0.07, 15.03±2.53 and 12.16±2.02 mg mL-1 (total protein), 0.28±0.012, 0.26±0.004, 0.20±0.035, 0.15±0.062, 0.11±0.036, 0.07±0.036 and 0.04±0.013 mg mL-1 (glucose), 72.65±7.88, 64.38±6.40, 64.24±4.80, 62.84±7.20, 62.11±3.84, 49.94±2.78, 38.31±3.29 IU L-1 (LDH) and 8.77±0.05, 4.87±0.25, 4.69±0.29, 4.37±0.02, 4.16±0.17, 2.87±0.29 and 2.83±0.26 mg mL-1 (total lipid). There were significant differences in sperm viability, total protein, glucose, LDH and total lipid between cryopreserved periods (p<0.05). As a conclusion, the sperm viability and biochemical changes of total protein, glucose, LDH and total lipid were determined in the cryopreserved sperm for control, 6, 12, 24 h, 7, 30 and 60 days. The storage duration in P. polyphagus cryopreserved sperm was also determined until 60 days and suitable for further breeding program.