In recent years it has become evident that lipoproteins play crucial roles in the assembly of bacterial envelope-embedded nanomachineries and in the processes of protein export/secretion. In this chapter we describe a method to determine their precise localisation, for example inner versus outer membrane, in Gram-negative bacteria using human opportunistic pathogen Pseudomonas aeruginosa as a model. A fusion protein between a given putative lipoprotein and the red fluorescent protein mCherry must be created and expressed in a strain expressing cytoplasmic green fluorescent protein (GFP). Then the peripheral localisation of the fusion protein in the cell can be examined by treating cells with lysozyme to create spheroplasts and monitoring fluorescence under a confocal microscope. Mutants in the signal peptide can be engineered to study the association with the membrane and efficiency of transport. This protocol can be adapted to monitor lipoprotein localisation in other Gram-negative bacteria.