Multitransmembrane proteins are notoriously difficult to analyze. To date, rapid, and cost-efficient detection methods are lacking and only mass spectrometry-based systems allow reliable quantification of these proteins. Here, we present a novel type of sandwich immunoassay that is capable of sensitively detecting multidrug resistance protein 1 (MDR1), a prototypic 12-transmembrane-domains transporter. In a first assay step, complex samples are enzymatically fragmented into peptides as routinely done for mass spectrometry. A proteotypic peptide derived from MDR1 was chosen and antibodies targeting this peptide were used to build a sandwich immunoassay. Validation of the optimized assay showed good sensitivity, reproducibility and it allowed reliable quantification of MDR1; cross-validation by mass spectrometry demonstrated the applicability for routine analyses in clinical and pharmaceutical research. MDR1 was quantified in primary human renal cell carcinoma and corresponding normal tissue and down-regulation or expression loss was found in tumor tissue corroborating its importance in drug resistance and efficacy.