The higher complexity of the spinning mechanism in the Orbicularia clade is related to a more complex ecological and behavioral use of various specialized silks formed by the assembly of complex and specialized repertoire of spidroin proteins. Actinopus spp. utilizes its primitive web mainly to cover burrows made in the ground, used for shelter and hunting. On the other hand, G. cancriformis is capable of building complex flat spiral webs and uses the web for a variety of behaviors, such as building the web’s radial support; filling the spiral part of the web; going down from trees; wrapping insects; making a sticky glue; etc. GenBank contains only 2 single and partial sequences for the entire Actinopus genus. Nineteen partial sequences have been deposited for the genus Gasteracantha, and most of them code for ribosomal RNAs, histones and cytochrome oxidase genes. For G. cancriformis, there are 15 sequenced genes, including the one encoding the major ampullate spidroin 2. The entire order Araneae has fewer than 29,000 sequences deposited in GenBank, including the dbEST database. Until now, the broadest analysis of spider transcriptomes involved the Mygalomorphae family Theraphosidae, better known as tarantulas. In 2006, a group from the University of Sa?o Paulo sequenced and analyzed 7,584 transcripts from the hemocytes of the tarantula spider Acanthoscurria gomesiana, characterizing about 1,500 new genes in this organism. Using gene ontology for transcript annotation, they identified an abundance of cDNAs for hemocyanin, lectin, structural constituents of ribosomes and the cytoskeleton, as well as 73 transcripts possibly involved in the spider immune response. In 2009, 2,507 59 ESTs from the skeletal muscle of another tarantula of the genus Aphonopelma were produced and analyzed. As expected, a significant number of skeletal muscle-related genes were found in their analysis, which supported the existence of both actin- and myosin-linked regulation of muscle contractions in the tarantula. Here, the cDNA repertoire obtained from Actinopus spp. and G. cancrifomis spinning glands were evaluated under a strict bioinformatics methodology. Therefore, the present work represents the most extensive characterization of spider transcriptomes to date, describing 78,913 transcriptomic sequences from the spinning glands of Actinopus spp. and G. cancriformis, thus increasing over 2.5fold the number of spider sequences available in public databases. Because Benzethonium Chloride sequence assembly is known to be influenced by the informational content of sequencing reads, we have tested the assembly of high-quality sequencing reads using three well-known software for EST assembly. A careful evaluation of the results was conducted before choosing the best dataset to go further on sequence characterization and similarity measures. Internal consistence metrics based on ICI index depicted from BLAST searches were taken on account to evaluate appropriate read to contig mapping. Bigger ICI values Mepiroxol represent better consensus built based on reads’ complete sequence and higher percent identity on HSP hits. The worst ICI value was shown by Celera assembly software, while CAP3 and MIRA presented better performances on ICI tests for both datasets. A subset of reads clustered in contigs by all the three software tested have had their ICI scores averaged and once again CAP3 have shown the best results on their assembly. In a final check on internal consistence we counted the number of reads having absolutely no BLAST hits against their associated consensus sequences.