The tear fluid is the aqueous layer of the thin trilaminar tear film which has a role of defence of the ocular surface. We first explored its protein composition with different proteomics techniques in order to assess the best protocol to use with this type of sample and to improve the list of proteins found in tears. Secondly, we applied this protocol to find new biomarkers for Multiple Sclerosis (MS) disease. Two 1D electrophoresis and three Off-gel electrophoresis were performed with a tryptic digestion and samples were analysed using two mass spectrometers. The isobaric Tandem Mass Tag (TMT) technology was used to label digested TF extracts from control and MS patients. Globally, combining these proteomics strategies, we were able to identify 399 TF proteins. 51 differentially expressed proteins were identified between control and MS patients. Our data demonstrate our ability to improve this biological fluid proteome by providing more than 100 new proteins. Moreover, some interesting proteins were showed to be differentially expressed in the TFs between control and MS patients.