To explore myringosclerotic tissue levels of two bone modeling markers: osteopontin (OPN) and osteoprotegerin (OPG) in experimentally-induced, histologically-confirmed myringosclerosis (MS). Materials & Methods: The right middle ear of 24 normal healthy growing male Wister rats was inoculated, via transtympanic access, by Streptococcus pneumoniae type 3, after a period of 8 weeks the tympanic membrane (TM) was examined Otomicroscopically for transparency and graded as normal TM, mild or marked opacification. Then, tympanic bullae were removed and a part of myringosclerotic plaque was excised for ELISA estimation of tissue extract levels of OPN and OPG and the remainder of the TM was stained with hematoxylin-eosin for light microscopic grading of TM inflammation according to extent of calcification into 5 grades. Results: 20 ears developed otoscopically defined myringosclerotic changes, 14 ears showed mild and 6 had marked opacification that was localized in 4 ears and diffuse in 2 ears. Histological examination reported inflammation of grade III in 7, grade IV in 13 and grade V in 4 specimens. Mean estimated tissue extract level of OPN and OPG in studied animals were significantly higher compared to control animals with a positive significant correlation between histological grading and tissue-extract levels of both OPG and OPN and a positive significant correlation between otoscopic grading and tissue-extract levels of OPG, but the correlation was non-significant with OPN Conclusion: Increased myringosclerotic tissue extract levels of both bone modeling markers indicated their possible role in initiation and/or progression of sclerotic changes in TM after chronic suppurative otitis media. [Ahmed Al-Shal, Nahla E. El-Azab, Mohamed Khaled Mohamed Mahfouz. Biochemical Determination of Tissue Ossification Markers in Experimentally-induced Myringosclerosis.