The stability and conformation of tripalmitin (TP) and triolein (TO) Langmuir-Blodgett (LB) monolayers on glass were investigated using Atomic Force Microscopy (AFM). Compression isotherms revealed that TP reached the solid state at 15 mN m−1, with a trident conformation. LB deposition of the monolayer on the glass surface was performed at the solid state (30 mN m−1) with a transfer ratio close to 1. The TO monolayer collapsed in the liquid-expanded phase at 13 mN m−1. LB deposition was performed at 11 mN m−1 with a transfer ratio of 1.4. The LB films were imaged by AFM (tapping mode). The TO monolayer collapsed in amorphous droplets. TP presented tiny amorphous drops plus crystalline shapes with unit layers of a thickness of 40—46 Å, corresponding to the long spacing of the β and α polymorphic forms. TP monolayer thus collapsed in aggregates to form crystalline structures. The thickness of the first step was mostly 86 Å, but sometimes 68 Å steps were observed, while the transferred monolayer generally reorganizes as a tuning fork in a regular crystalline unit, but can also remain as a trident, providing a hydrophobic support for upper layers. This findings supply backing for technical processes such as the interpretation of fat fractionation by microfiltration membranes, based on triglyceride/solid interaction statements.