Ischemic heart disease is the leading cause of deaths worldwide. Thus, understanding the molecular mechanisms underlying disease progression is needed. Due to heart importance and lack of studies evaluating different sample preparation methods for heart proteomics, we compared three well-established protocols in shotgun proteomics using dimethyl label quantitation to allow relative quantitation. The tested methods for the analysis of left ventricle (LV) tissue were: i) in-solution digestion (ISD); ii) on-pellet digestion (OPD); and iii) on-filter digestion (OFD). Protein extraction was done using SDS-containing buffer for OPD and OFD while this step was under urea-containing buffer for ISD. We used an optimized one-step reaction for reduction of disulfide bonds and alkylation of thiol groups in ISD and OPD. Using the same amount of tissue, we observed that OFD and ISD extracted significantly higher amount of protein than OPD. ISD outperformed OFD and OPD in the number of proteins identified. We did not observe significant bias related to physicochemical features of the identified proteins when comparing the three protocols. ISD was more efficient to identify low abundant proteins and yielded more proteins per protocol duration. Thus, we concluded that the optimized ISD suited better for heart proteomics than OFD and OPD.