Six methods for extraction of surface-associated proteins were applied to Lactobacillus rhamnosus GG (LGG) cells harvested at early stationary phase. The six separation profiles of proteins analysed by gel electrophoresis were similar but markedly different from total protein extracts, suggesting that those profiles contained surface-associated proteins and were not contaminated by cytoplasmic proteins. Among the methods tested, treatment with 5 m LiCl appeared to be the most suitable for surface-associated protein extraction in this probiotic strain, since it allowed a maximum recovery of proteins. Among the proteins identified, ribosomal proteins, cytoplasmic proteins already described on the bacterial surface, and several proteins predicted to be surface-associated were identified by tandem mass spectrometry. The pH of the buffer affected the efficiency of protein extraction, with less surface proteins being extracted from LGG cells at acidic pH. Some of the surface proteins identified in this work may help explain some of the probiotic traits of LGG, notably these concerning the interaction of LGG strain with the human host.