Abstract The aim of this study was to synthesize simple thiol-reactive conjugates from maleimide and lipoamines (stearylamine or oleylamine) and to develop a simple, fast and low-cost method for the preparation of lyophilized general-purpose thiol-reactive liposomes. A formulation of egg phosphatidylcholine-dipalmitoylphoshatidylglycerol (9:0.1 molar ratio) was developed and characterized. Freeze-drying methodology was established to produce a stock of liposomes and the physicochemical characteristics of the reconstituted liposomes were compared with those of the initial preparation. The physicochemical properties (size and ζ-potential) of the new liposomal formulations were studied. High-performance thin-layer chromatography coupled to a flame ionization detector was applied for one-step analysis of the liposomal components and for determining the maleimide-lipoamine conjugates phospholipid molar ratio. The differences concerning the incorporation efficiency of the synthetic conjugates into liposomes were discussed on the basis of their conformational properties. The small difference in structure between the two thiol-reactive conjugates (i.e., the C18 alkyl chain double bond) causes a considerable difference in phospholipids packing of the resulting lipidic bilayers of the liposomes; the conformational bending of conjugate maleimide-oleylamine may contribute to the final architecture of liposomes.