Cardiolipin (CL) is a uniquely dimeric phospholipid found exclusively in the inner mitochondrial membrane (IMM). Its presence is critically important for the shape of IMM structures and oxidative phosphorylation, the means by which mitochondria produce energy. Though there are many ways to study cardiolipin, studying it within a flat lipid bilayer is not an established method. Due to CL’s unique structure, it is generally difficult to incorporate it into straight phospholipid bilayers. Thus, we sought to study if membranes could be formed with a significant concentration of CL. We hypothesized that membrane dynamics, quantified as diffusion coefficient and mobile fraction, would be decreased with increasing incorporation of CL.We created membranes of 0, 15, and 30 mol% CL fluorescently labeled with Texas Red DHPE (TR-DHPE) with the remainder of lipids being 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). These membranes were each in buffers of phosphate and Tris at concentrations of 20 mM and pH 7.40. These membranes were assessed by their fluorescent recovery after photobleaching (FRAP). A small area on each membrane was subjected to intense light, causing its fluorescent emission to decrease. The photobleached phospholipids diffused with the fluorescently labeled ones over time, causing the spot to lighten. Analyzing these recoveries enabled us to quantify the diffusivity of each and what fraction of phospholipids are freely able to participate in diffusion. FRAP revealed that increasing the concentration of CL in POPC membranes led to less diffusivity. However, the decrease in diffusivity was larger in the Tris-buffered membranes, suggesting that the Tris buffer impedes phospholipid diffusion in the presence of CL. As for the mobile fraction, some membranes displayed bizarre values greater than 1. This was explained by over-recovery due to the interplay between self-quenching and photobleaching. The findings about mobile fraction values were most peculiar with the Tris membranes, demonstrating that Tris may cause more than one issue in model membrane evaluation.Our results indicate that the buffer environment might have a significant effect on membrane dynamics. However, further research is required to ascertain what kinds of buffer attributes give rise to such an effect.