Malaria parasites are able to alter the number of male and female gametocytes produced, thereby changing the sex ratio of the reproductive stages. Alteration of the sex ratio has been suggested to increase the reproductive oputput under different conditions, thereby maximizing the transmission success and influencing the epidemiology of this disease. The only existing method for quantifying sex ratios of P. Jalciparum is based on the visual identification of male and female gametocytes by light microscopy. However, this method is limited, as it only allows studying microscopically detectable gametocyte densities found in natural infections and parasites at sub-microscopic levels are not accounted for. A new and more accurate method for estimating sex ratios of P. Jalciparum, which includes large sample sizes obtained by magnetic purification, is described here. In this study light microscopy, immunofluorescent antibody detection (IFA) and quantitative Real-Time PCR (qRT-PCR) were used to estimate sex ratios in P. Jalciparum. The qRT-PCR assay allowed measurement of mRNA expression levels for sexspecific and sexual stage proteins in a multiplex analysis. The female specific protein Pfg377 (PFL2405c) and the putative male specific protein Alpha-tubulin II (PFD1050w) were used to quantify either female or male gametocytes, with Pfs16 (pFD0310w) and Pfs25 (PFlO_0303) serving to quantify the total number of gametocytes present. Results suggest that Alpha-tubulin II is not in fact a male specific protein, whereas Pfg377 was found to be only present in female gametocytes. Different patterns of gene expression during gametocytogenesis have been identified for the four key sexual stage genes in a multiplex assay. Relative expression data of Pfg377 versus Pfs 16 or Pfs25 reveal that the quantification of sex ratios from small volumes of gametocyte-positive blood is possible, and should be applicable for using finger-prick peripheral blood samples from gametocyte carriers.