jats:pSum Frequency Generation (SFG) spectroscopy is widely used for studying the di-chain phospholipid monolayers incorporated in model cell membranes. In this context, it is frequently assumed, without justification, that the chains are identical, so their individual contributions to the SFG spectra are indistinguishable. However, the combination of both attractive and repulsive Van der Waals interactions between the chains results in a finite angle between their two terminal methyl groups, resulting in non-equivalent contributions to the non-linear susceptibility. This work describes the application of the underlying non-linear theory required to produce the accurate SFG spectral simulations needed to test this assertion and therefore provides the necessary quantitative validation. For phospholipids comprising two identical saturated chains, which typically have small angles of divergence, these simulations predict only small deviations in the SFG intensities from those calculated assuming a single methyl orientation. Non-identical tails, however, with differences in the degree or type of chain unsaturation, or in the parity of the chain lengths, show much larger discrepancies than the assumption of a single chain. In these cases, the two tails must be treated as separate entities, and their structural relationship must be incorporated into the interpretation of their SFG spectra. A second important result from the simulations arises from the systematic nature of the deviations, which shows that even small intensity changes should not be quickly dismissed on the basis of being subsumed by the uncertainties associated with spectral noise.</jats:p>