Updated project metadata. Leishmania survival inside insect and mammalian hosts depends on the parasite’s capacity to develop into promastigote and amastigote stages, respectively. Promastigotes can be maintained in vitro in culture medium mimicking the insect milieu in which the parasites are surviving, differentiating and proliferating. We previously showed that the maintenance of the parasites in this in vitro environment lead to a progressive loss of infectivity together with genomic changes, notably chromosome amplifications. In contrast to classical eukaryotes Leishmania do not regulate expression of protein coding genes by transcriptional control, thus ruling out regulated gene expression changes in adaptation. In the absence of classical transcriptional regulation, Leishmania has evolved and emphasized other forms of control that are relevant for evolutionary adaptation, notably regulation of RNA abundance by gene dosage variation. In order to identify the biological signals that correlate with the observed fitness trade off we observed during L. donovani culture adaptation we combined DNAseq and RNAseq with label-free quantitative proteomics. Promastigotes freshly derived from splenic amastigotes purified from the spleen of infected hamsters were maintained in culture for 2 (EP) and 20 (LP) passages. Total protein extracts from four EP and four LP promastigotes were prepared for label free MS analysis.