Updated project metadata. Purpose: The identification of protein isoforms in complex biological samples is challenging. We, therefore, used a mass spectrometry (MS) approach to unambiguously identify cardiac myofilament protein isoforms based on the observation of a tryptic peptide consisting of a sequence unique to a particular isoform. Experimental design: Three different workflows were used to isolate and fractionate the rat cardiac myofilament subproteomes. All fractions were analyzed on an LTQ-Orbitrap MS, proteins were identified using various search engines (Mascot, X!Tandem, X!Tandem Kscore and OMSSA) with results combined via PepArML Meta-Search Engine, and a post-search analysis performed by MASPECTRAS. Results: The combination of multiple workflows and search engines resulted in a larger number of non-redundant proteins identified than individual methods. A total of 461 proteins were observed overlapping in two or three of the workflows. Literature search for myofilament presence with manual validation of the MS spectra was carried out for unambiguous identification: 10 cardiac myofilament and 17 cardiac myofilament-associated proteins were confirmed to have multiple isoforms or sub-isoforms. Conclusion and clinical relevance: We have identified multiple isoforms of myofilament proteins that are present in cardiac tissue using unique tryptic peptides. Changes of distribution of these protein isoforms under pathological conditions could ultimately allow for clinical diagnostics or as therapeutic targets.