Absolute quantification of peptides by mass spectrometry requires a reference, frequently using heavy isotope coded peptides as internal standards. These peptides have traditionally been generated by chemical stepwise synthesis. Recently, a new way to supply such peptides was described in which nucleotide sequences coding for the respective peptides are concatenated into a synthetic gene (QconCAT). This gene is then expressed and digested to yield desired peptides. Even though both of these methods for peptide production are routinely used for absolute quantifications, there is currently no information regarding the accuracy of the quantifications made in each case. In this report, we used sets of synthetic and biological peptides in parallel to evaluate the accuracy of either method. Twenty five peptides derived from the C. elegans proteome were selected for this study. Twenty four were successfully chemically synthesized. Five QconCAT genes were designed, each a concatenation of the same 25 peptides, but each in separate, different randomized order, and expressed via in vitro translation reactions that contained heavy isotope labeled lysine and arginine. Three of the five QconCATs were successfully produced. All three QconCAT polypeptides were then digested using the optimized conditions, and then mixed in a 1:1 ratio with their synthetic counterparts. Multi-reaction monitoring (MRM) mass spectrometry was then used for quantification. Results showed that the digestion protocol had a significant impact on equimolarity of final peptides, confirmed the need for optimization. Under optimal conditions, however, most QconCAT peptides were produced at an equimolar ratio. A few QconCAT-derived peptides were largely overestimated due to problems with solubilization, or stability of the synthetic peptides. Overall, neither the chemical synthesis nor the recombinant genetic approach proved to be superior as a method for the production of reference peptides for absolute quantification.