The backscatter coefficient (BSC) has demonstrated the ability to classify disease state and to monitor changes in tissue due to therapy. However, 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 tissue region to be interrogated. The intervening layers result in increases in bias and variance of BSC-based estimates. To accurately account for the transmission losses, an in situ calibration approach is proposed to obtain a more robust estimate of the BSC. The in situ calibration technique employs the use of a biocompatible sphere that is well-characterized ultrasonically and embedded inside a sample or tissue. Ultrasound scattered from the sphere encounters the same transmission loss and attenuation as the investigated sample and can be used as a reference spectrum to provide a more accurate estimate of the BSC. A 2-mm diameter titanium sphere was embedded inside a homogeneous phantom containing glass bead scatterers with diameters ≤ 90 μm placed spatially at random. A layer of fatty meat was placed on top of the phantom to produce transmission losses from a layer. The BSC was estimated from the phantom with and without the layer on top and compared using a traditional reference phantom technique and using the in situ sphere as a calibration target. Estimates of the effective scatterer diameter (ESD) were obtained for each condition and compared. The BSCs estimated using the in situ calibration approach with and without the layer present overlapped with the BSC estimated using the traditional reference phantom approach without the layer present. The BSC estimated using the traditional reference phantom approach with the layer present did not overlap with the other curves. Estimates of the ESD were 80 μm, 82 μm, 84 μm and 95 μm using the in situ calibration approach without the layer, with the layer and using the reference phantom approach without layer and with the layer present, respectively. The results indicate that an in situ calibration target can account for overlying tissue losses thereby improving the robustness of BSC-based estimates. This work was supported by a grant from the NIH (R21 EB020766).