A comparative analysis of protein extracted from different regions of silk glands in the Bombyx mori L. and Philosamia ricini Hutt was performed employing single-dimensional-electrophoresis technique. Notably, a protein extracted directly from the lumen of the middle silk gland yielded two discrete protein bands with molecular mass of 325 and 26 kDa representing fibroin heavy (H) and low (L) chains than whole silk gland of B. mori. Contrastingly, such differentiation in protein separation could not be achieved due to undifferentiated regions of silk gland in P. ricini. Interestingly, copious amount of proteins with different molecular weight were noticed in the whole silk gland of B. mori and P. ricini, whose functional properties in the silk gland remain enigmatic. Thus, middle and whole silk glands of B. mori shall be an ideal source for the extraction of fibroin and sericin respectively as potent bio-materials for biotechnological and biomedical applications.