The microstructural changes of semicrystalline bivoltine silk fibers annealed between 27 to 260 degrees C were measured using positron annihilation lifetime spectroscopy (PALS). Measured lifetime and intensity as a function of temperature reveals the removal of water molecules until 60 degrees C. Present results indicate a T-g of 140 degrees C, which is further confirmed by differential scanning calorimetry (DSC). The structural changes like random-coil to alpha and then to beta form are observed in the crystalline and amorphous domains at 100 and 205 degrees C, respectively. Degradation of the fibers occur around 240 degrees C. As an extension of the present measurements, we have estimated the activation energy of 8.0 KJ/mol in the disordered domain of the fiber, based on Goldanskii’s kinetic relations.