“Immuno-flowFISH” is a world-first automated high-throughput imaging flow cytometry method that can detect numerical and structural abnormalities in phenotypically identified neoplastic cells. This method was successfully applied to the identification of trisomy12 and del(17p) in chronic lymphocytic leukaemia. In this study, we aimed to expand the method to assess reciprocal chromosomal translocations that guide therapeutic decision-making in Multiple Myeloma (MM). Briefly, nucleated cells were isolated from the blood of healthy adults or blood/bone marrow of MM patients (n = 8). Immunophenotyping was performed with fluorescently conjugated CD138, CD38, CD45 and CD19 monoclonal antibodies. Following fixation, cells were permeabilised, double-stranded DNA denatured and hybridised with chromosome 12 (control) or dual fusion MM translocation FISH probes (t(4;14)/IGH-FGFR3, t(11;14)/IGH-MYEOV). Cells were analysed on the Amnis ImageStreamX Mark II and data analysed to determine the percent of cells positive for each immunophenotype, FISH spot count and presence of translocation. Method optimisation included evaluation of different combinations of fluorophores conjugated to antibodies and probes; hybridisation conditions; probe concentration and sample acquisition (e.g. laser excitation and compensation). To date, we have established a diagnostic MM immunophenotyping panel suitable for FISH analysis consisting of CD138-V500c, CD38-BV480, CD45-BV605 and CD19-AF647 antibodies that can reliably identify clonal neoplastic cells (CD138+/CD38+/CD45+dim/CD19-) concordant with diagnostic flow cytometry (2.04 – 77.7%). Increases in laser excitation input (488nm & 561nm) with appropriate data compensation, utilisation of Green (521nm), Orange (572nm) and Texas-Red (610nm) fluorescence emitting conjugates and increases in probe to cell concentration (2-8x) per test improved mean fluorescence intensity for spot-counting by software enumeration and visualisation by 2-3 fold (80%+ cells with detectable IGH-FGFR3 FISH signals). Further improvements to the immuno-flowFISH protocol such as FISH hybridisation and optimisation of software analysis (i.e. detection of true-positive dual fusion IGH-FGFR3/MYEOV events) may further provide a robust assessment of MM suitable for enhanced clinical utility.