Background: The outermost layer of the tear film consists of a thin lipid layer (LL). The lipid layer serves as a barrier against evaporation of the aqueous component of the tear film. The ability to simultaneously image both the lipid layer thickness and overall tear film thickness is novel, and will help further understandings of mechanisms of how the lipid layer assembles and interacts with the full tear film thickness. Methods: We developed a system that combines simultaneous optical coherence tomography (OCT) and thickness dependent fringes (TDF) interferometry for in vivo imaging of the tear film. The OCT possesses an axial resolution of 1.38 μm in tear film, providing an accurate measurement of the thickness of the overall tear film. The TDF can detect a minimal change of approximately 15 nm in LL thickness. In addition, the spatial resolution of TDF images in x-y plane is 5 μm. Results: The effect of instilling artificial tears on the PCTF and PLTF was examined. In both contact lens and non-contact lens wear, it could be observed from the OCT results that instillation of artificial tears increased the thickness of the overall tear film immediately, followed by a gradual reduction thereafter. These findings were consistent with other studies. However, unlike those previous reports, the thickness of the LL in this study was quantified simultaneously with the TDF subsystem. The results showed that bulking agents such as these artificial tears were not necessarily intended to increase the LL thickness. Immediately after instillation of artificial tears, the PCTF increased from 4.4 ± 0.97 to 20.3 ± 3.6 μm. The PCTF then decreased to 8.8 ± 2.1 μm at 4 min post-instillation. The thicknesses of the LL were 62.4 ± 14.5 nm, 48.7 ± 5.3 nm, and 55.2 ± 9.8 nm at pre-drop instillation, post-drop instillation, and 4-min post-drop instillation, respectively. Conclusions: In this work, we have described a novel imaging system that integrated OCT and TDF imaging techniques, which may facilitate the study of many physiological and clinical aspects of the tear film.