The objective of this article is to demonstrate the feasibility of estimating the backscatter coefficient (BSC) using an in situ calibration source. Traditional methods of estimating the BSC in vivo using a reference phantom technique do not account for transmission losses due to intervening layers between the ultrasonic source and the tissue region to be interrogated, leading to increases in bias and variance of BSC-based estimates. To account for transmission losses, an in situ calibration approach is proposed. The in situ calibration technique employs a titanium sphere that is well-characterized ultrasonically, biocompatible, and embedded inside the sample. A set of experiments was conducted to evaluate the embedded titanium spheres as in situ calibration targets for BSC estimation. The first experiment quantified the backscattered signal strength from titanium spheres of three sizes: 0.5, 1, and 2 mm in diameter. The second set of experiments assessed the repeatability of BSC estimates from the titanium spheres and compared these BSCs to theory. The third set of experiments quantified the ability of the titanium bead to provide an in situ reference spectrum in the presence of a lossy layer on top of the sample. The final set of experiments quantified the ability of the bead to provide a calibration spectrum over multiple depths in the sample. All experiments were conducted using an L9-4/38 linear array connected to a SonixOne system. The strongest signal was observed from the 2-mm titanium bead with the signal-to-noise ratio (SNR) of 11.6 dB with respect to the background speckle. Using an analysis bandwidth of 2.5-5.5 MHz, the mean differences between the experimentally derived BSCs and BSCs derived from the Faran theory were 0.54 and 0.76 dB using the array and a single-element transducer, respectively. The BSCs estimated using the in situ calibration approach without the layer and with the layer and using the reference phantom approach with the layer were compared to the reference phantom approach without the layer present. The mean differences in BSCs were 0.15, 0.73, and -9.69 dB, respectively. The mean differences of the BSCs calculated from data blocks located at depths that were either 30 pulse lengths above or below the actual bead depth compared to the BSC calculated at bead depth were -1.55 and -1.48 dB, respectively. The results indicate that an in situ calibration target can account for overlaying tissue losses, thereby improving the robustness of BSC-based estimates.